Chloride potassium symporter 5

Potassium-chloride transporter member 5 (aka: KCC2 and SLC12A5) is a neuron-specific chloride potassium symporter responsible for establishing the chloride ion gradient in neurons through the maintenance of low intracellular chloride concentrations. It is a critical mediator of synaptic inhibition, cellular protection against excitotoxicity and may also act as a modulator of neuroplasticity. Potassium-chloride transporter member 5 is also known by the names: KCC2 (potassium chloride cotransporter 2) for its ionic substrates, and SLC12A5 for its genetic origin from the "SLC12A5" gene in humans.
Animals with reduced expression of this transporter exhibit severe motor deficits, epileptiform activity, and spasticity. KCC2 knockout animals, in which KCC2 is completely absent, die postnatally due to respiratory failure.
Location.
KCC2 is a neuron-specific membrane protein expressed throughout the central nervous system, including the hippocampus, hypothalamus, brainstem, and motoneurons of the ventral spinal cord.
At the subcellular level, KCC2 has been found in membranes of the somata and dendrites of neurons, with no evidence of expression on axons. KCC2 has also been shown to colocalize with GABAA receptors, which serve as ligand-gated ion channels to allow chloride ion movement across the cell membrane. Under normal conditions, the opening of GABAA receptors permits the hyperpolarizing influx of chloride ions to inhibit postsynaptic neurons from firing.
Counterintuitively, KCC2 has also been shown to colocalize at excitatory synapses. One suggested explanation for such colocalization is a potential protective role of KCC2 against excitotoxicity. Ion influx due to the excitatory synaptic stimulation of ion channels in the neuronal membrane causes osmotic swelling of cells as water is drawn in alongside the ions. KCC2 may help to eliminate excess ions from the cell in order to re-establish osmotic homeostasis.
Structure.
KCC2 is a member of the cation-chloride cotransporter (CCC) superfamily of proteins.
As with all CCC proteins, KCC2 is an integral membrane protein with 12 transmembrane domains and both N- and C-terminal cytoplasmic domains. The terminal cytoplasmic domains can be phosphorylated by kinases within the neuron for rapid regulation.
Two Isoforms: KCC2a, KCC2b.
There are two isoforms of KCC2: KCC2a and KCC2b. The two isoforms arise from alternative promoters on the "SLC12A5" gene and differential splicing of the first mRNA exon. The isoforms differ in their N-termini, with the KCC2a form constituting the larger of the two splice variants.
KCC2a levels remain relatively constant during pre- and postnatal development.
KCC2b, on the other hand, is scarcely present during prenatal development and is strongly upregulated during postnatal development. The upregulation of KCC2b expression is thought to be responsible for the “developmental shift” observed in mammals from depolarizing postsynaptic effects of inhibitory synapses in early neural networks to hyperpolarizing effects in mature neural networks.
KCC2b knockout mice can survive up to postnatal day 17 (P17) due to the presence of functional KCC2a alone, but they exhibit low body weight, motor deficits and generalized seizures. Complete KCC2 knockouts (both KCC2a and KCC2b absent) die after birth due to respiratory failure.
Oligomerization.
Both KCC2 isoforms can form homomultimers, or heteromultimers with other K-Cl symporters on the cell membrane to maintain chloride homeostasis in neurons. Dimers, trimers, and tetramers involving KCC2 have been identified in brainstem neurons. Oligomerization may play an important role in transporter function and activation, as it has been observed that the oligomer to monomer ratio increases in correlation to the development of the chloride ion gradient in neurons.
Developmental changes in expression.
KCC2 levels are low during mammalian embryonic development, when neural networks are still being established and neurons are highly plastic (changeable). During this stage, intracellular chloride ion concentrations are high due to low KCC2 expression and high levels of a transporter known as NKCC1 (Na+/K+ chloride cotransporter 1), which moves chloride ions into cells. Thus, during embryonic development, the chloride gradient is such that stimulation of GABAA receptors and glycine receptors at inhibitory synapses causes chloride ions to flow out of cells, making the internal neuronal environment less negative (i.e. more depolarized) than it would be at rest. At this stage, GABAA receptors and glycine receptors act as excitatory rather than inhibitory effectors on postsynaptic neurons, resulting in depolarization and hyperexcitability of neural networks.
During postnatal development, KCC2 levels are strongly upregulated while NKCC1 levels are down regulated. This change in expression correlates to a developmental shift of the chloride ion concentration within neurons from high to low intracellular concentration. Effectively, as the chloride ion concentration is reduced, the chloride gradient across the cellular membrane is reversed such that GABAA receptor and glycine receptor stimulation causes chloride ion influx, making the internal neuronal environment more negative (i.e. more hyperpolarized) than it would be at rest. This is the developmental shift of inhibitory synapses from the excitatory postsynaptic responses of the early neural development phase to the inhibitory postsynaptic responses observed throughout maturity.
Function.
Current literature suggests that KCC2 serves three primary roles within neurons:
Postsynaptic inhibition.
KCC2 is a potassium (K+)/chloride (Cl−) symporter that maintains chloride homeostasis in neurons. The electrochemical chloride gradient established by KCC2 activity is crucial for classical postsynaptic inhibition through GABAA receptors and glycine receptors in the central nervous system. KCC2 utilizes the potassium gradient generated by the Na+/K+ pump to drive chloride extrusion from neurons. In fact, any disruption of the neuronal K+ gradient would indirectly affect KCC2 activity.
Loss of KCC2 following neuronal damage (i.e. ischemia, spinal cord damage, physical trauma to the central nervous system) results in the loss of inhibitory regulation and the subsequent development of neuronal hyperexcitability, motor spasticity, and seizure-like activity as GABAA receptors and glycine receptors revert from hyperpolarizing to depolarizing postsynaptic effects.
Cellular protection.
High levels of stimulation and subsequent ionic influx through activated ion channels can result in cellular swelling as osmotically-obliged water is drawn into neurons along with ionic solutes. This phenomenon is known as excitotoxicity. KCC2 has been shown to be activated by cell-swelling, and may therefore play a role in eliminating excess ions following periods of high stimulation in order to maintain steady-state neuronal volume and prevent cells from bursting.
This role may also account for the fact that KCC2 has been known to colocalize near excitatory synapses, even though its primary role is to establish the chloride gradient for postsynaptic inhibition.
Morphogenesis and function of glutamatergic synapses.
In addition to controlling the efficacy of GABAergic synapses through chloride homeostasis, KCC2 play a critical role in the morphogenesis and function of glutamatergic synapses within the central nervous system. Studies on hippocampal tissue in KCC2 knockout animals showed that neurons lacking KCC2 have stunted dendritic growth and malformed dendritic spines. Recent studies demonstrate that KCC2 plays a critical role in the structure and function of dendritic spines which host most excitatory synapses in cortical neurons. Through an interaction with actin cytoskeleton, KCC2 forms a molecular barrier to the diffusion of transmembrane proteins within dendritic spines, thereby regulating the local confinement of AMPA receptors and synaptic potency.
It has been proposed that the downregulation of KCC2 observed following neuronal trauma, and the consequent depolarizing shift of GABAA-mediated synapses, may be an aspect of neuronal de-differentiation. De-differentiation of damaged portions of the nervous system would allow for neuronal networks to return to higher levels of plasticity in order to rewire surviving neurons to compensate for damage in the network. In addition, reduced glutamatergic transmission upon KCC2 downregulation may serve as a homeostatic process to compensate for the reduced GABA transmission due to altered chloride extrusion.
Oncogenesis.
Mutations in SLC12A5 are associated with colon cancer.
Regulation.
Transcriptional regulation: TrkB receptor signalling.
KCC2 is transcriptionally downregulated following central nervous system injury by the TrkB receptor signalling transduction cascade (activated by BDNF and NT-4/5).
Post-translational regulation: phosphorylation.
It is conventionally thought that phosphorylation inactivates or downregulates KCC2, however there is recent evidence to suggest that phosphorylation at different sites on the KCC2 protein determines different regulational outcomes:
KCC2 has an extremely high rate of turnover at the plasmalemma (minutes), suggesting that phosphorylation serves as the primary mechanism for rapid regulation.
Activity-dependent downregulation.
KCC2 is downregulated by excitatory glutamate activity on NMDA receptor activity and Ca2+ influx. This process involves rapid dephosphorylation on Ser940 and calpain protease cleavage of KCC2, leading to enhanced membrane diffusion and endocytosis of the transporter, as demonstrated in experiments using single particle tracking.
Glutamate release occurs not only at excitatory synapses, but is also known to occur after neuronal damage or ischemic insult. Thus, activity-dependent downregulation may be the underlying mechanism by which KCC2 downregulation occurs following central nervous system injury.


Methylfluorophosphonylcholine

Methylfluorophosphonylcholine (MFPCh) is an extremely toxic chemical compound related to the G-series nerve agents. It is an extremely potent acetylcholinesterase inhibitor which is around 100 times more potent than sarin at inhibiting acetylcholinesterase "in vitro", and around 10 times more potent "in vivo", depending on route of administration and animal species tested. MFPCh is resistant to oxime reactivators, meaning the acetylcholinesterase inhibited by MFPCh can't be reactivated by oxime reactivators. MFPCh also acts directly on the acetylcholine receptors. However, despite its high toxicity, methylfluorophosphonylcholine is a relatively unstable compound and degrades rapidly in storage, so it was not deemed suitable to be weaponised for military use.


Fibulin

Fibulin (FY-beau-lin) (now known as Fibulin-1 FBLN1) is the prototypic member of a multigene family, currently with seven members. Fibulin-1 is a calcium-binding glycoprotein. In vertebrates, fibulin-1 is found in blood and extracellular matrices. In the extracellular matrix, fibulin-1 associates with basement membranes and elastic fibers. The association with these matrix structures is mediated by its ability to interact with numerous extracellular matrix constituents including fibronectin, proteoglycans, laminins and tropoelastin. In blood, fibulin-1 binds to fibrinogen and incorporates into clots.
Fibulins are secreted glycoproteins that become incorporated into a fibrillar extracellular matrix when expressed by cultured cells or added exogenously to cell monolayers. The five known members of the family share an elongated structure and many calcium-binding sites, owing to the presence of tandem arrays of epidermal growth factor-like domains. They have overlapping binding sites for several basement-membrane proteins, tropoelastin, fibrillin, fibronectin and proteoglycans, and they participate in diverse supramolecular structures. The amino-terminal domain I of fibulin consists of three anaphylatoxin-like (AT) modules, each approximately 40 residues long and containing four or six cysteines. The structure of an AT module was determined for the complement-derived anaphylatoxin C3a, and was found to be a compact alpha-helical fold that is stabilized by three disulphide bridges in the pattern Cys14, Cys25 and Cys36 (where Cys is cysteine). The bulk of the remaining portion of the fibulin molecule is a series of nine EGF-like repeats.


Peptide methionine sulfoxide reductase

Peptide methionine sulfoxide reductase could stand for:


2,5-dihydroxypyridine 5,6-dioxygenase

In enzymology, a 2,5-dihydroxypyridine 5,6-dioxygenase () is an enzyme that catalyzes the chemical reaction
The 2 substrates of this enzyme are 2,5-dihydroxypyridine and O2, whereas its product is N-formylmaleamic acid.
This enzyme belongs to the family of oxidoreductases, specifically those acting on single donors with O2 as oxidant and incorporation of two atoms of oxygen into the substrate (oxygenases). The oxygen incorporated need not be derived from O2. It employs one cofactor, iron.
This enzyme participates in nicotinate and nicotinamide metabolism.
Nomenclature.
The systematic name of this enzyme class is 2,5-dihydroxypyridine:oxygen 5,6-oxidoreductase. Other names in common use include 2,5-dihydroxypyridine oxygenase, and pyridine-2,5-diol dioxygenase.


Degron

A degron is a portion of a protein that is important in regulation of protein degradation rates. Known degrons include short amino acid sequences, structural motifs and exposed amino acids (often Lysine or Arginine) located anywhere in the protein. In fact, some proteins can even contain multiple degrons. Degrons are present in a variety of organisms, from the N-degrons (see N-end Rule) first characterized in yeast to the PEST sequence of mouse ornithine decarboxylase. Degrons have been identified in prokaryotes as well as eukaryotes. While there are many types of different degrons, and a high degree of variability even within these groups, degrons are all similar for their involvement in regulating the rate of a protein's degradation. Much like protein degradation (see proteolysis) mechanisms are categorized by their dependence or lack thereof on Ubiquitin, a small protein involved in proteasomal protein degradation, Degrons may also be referred to as “Ubiquitin-dependent" or “Ubiquitin-independent".
Types.
Ubiquitin-dependent degrons are so named because they are implicated in the polyubiquitination process for targeting a protein to the proteasome. In some cases, the degron itself serves as the site for polyubiquitination as is seen in TAZ and β-catenin proteins. Because the exact mechanism by which a degron is involved in a protein's polyubiqutination is not always known, degrons are classified as Ubiquitin-dependent if their removal from the protein leads to less ubiquitination or if their addition to another protein leads to more ubiquitination.
In contrast, Ubiquitin-independent degrons are not necessary for the polyubiquitination of their protein. For example, the degron on IkBa, a protein involved in the regulation of the immune system, was not shown to be involved in ubiquitination since its addition to Green Fluorescent Protein (GFP) did not increase ubiquitination. However, a degron can only hint at the mechanism by which a protein is degraded and so identifying and classifying a degron is only the first step in understanding the degradation process for its protein.
Identification.
In order to identify a portion of a protein as a degron, there are often three steps performed. First, the degron candidate is fused to a stable protein, such as GFP, and protein abundances over time are compared between the unaltered protein and the fusion (as shown in green). If the candidate is in fact a degron, then the abundance of the fusion protein will decrease much faster than that of the unaltered protein. Second, a mutant form of the degron's protein is designed such that it lacks the degron candidate. Similar to before, the abundance of the mutant protein over time is compared to that of the unaltered protein (as shown in red). If the deleted degron candidate is in fact a degron, then the mutant protein abundance will decrease much slower than that of the unaltered protein. Recall that degrons are often referred to as “Ubiquitin-dependent” or “Ubiquitin-independent” The third step performed is often done after one or both of the previous two steps, because it serves to identify the Ubiquitin dependence or lack thereof of a previously identified degron. In this step, protein A and A’ (identical in every way except the presence of degron in A’) will be examined. Note that mutation or fusion procedures could be performed here, so either A is a protein like GFP and A’ is a fusion of GFP with the degron (as shown in green) or A’ is the degron's protein and A is a mutant form without the degron (as shown in Red.) The amount of Ubiquitin bound to A and to A’ will be measured. A significant increase in the amount of Ubiquitin in A’ as compared to A will suggest that the degron is Ubiquitin-dependent.


SIGLEC5

Sialic acid-binding Ig-like lectin 5 is a protein that in humans is encoded by the "SIGLEC5" gene. SIGLEC5 has also been designated CD170 (cluster of differentiation 170).


6-methylsalicylic-acid synthase

In enzymology, a 6-methylsalicylic-acid synthase () is a polyketide synthase that catalyzes the chemical reaction
The 4 substrates of this enzyme are acetyl-CoA, malonyl-CoA, NADPH, and H+, whereas its 5 products are 6-methylsalicylate, CoA, CO2, NADP+, and H2O.
This enzyme belongs to the family of transferases, specifically those acyltransferases transferring groups other than aminoacyl groups. The systematic name of this enzyme class is acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl-reducing, thioester-hydrolysing and cyclizing). Other names in common use include MSAS, and 6-methylsalicylic acid synthase.


Cofactor (biochemistry)

A cofactor is a non-protein chemical compound or metallic ion that is required for an enzyme's activity as a catalyst (a catalyst is a substance that increases the rate of a chemical reaction). Cofactors can be considered "helper molecules" that assist in biochemical transformations. The rates at which these happen are characterized in an area of study called enzyme kinetics. Cofactors typically differ from ligands in that they often derive their function by remaining bound.
Cofactors can be divided into two types: inorganic ions and complex organic molecules called coenzymes. Coenzymes are mostly derived from vitamins and other organic essential nutrients in small amounts. (Note that some scientists limit the use of the term "cofactor" to inorganic substances; both types are included here.)
Coenzymes are further divided into two types. The first is called a "prosthetic group", which consists of a coenzyme that is tightly or even covalently, and permanently bound to a protein. The second type of coenzymes are called "cosubstrates", and are transiently bound to the protein. Cosubstrates may be released from a protein at some point, and then rebind later. Both prosthetic groups and cosubstrates have the same function, which is to facilitate the reaction of enzymes and proteins. An inactive enzyme without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is called a holoenzyme. (Note that the International Union of Pure and Applied Chemistry (IUPAC) defines "coenzyme" a little differently, namely as a low-molecular-weight, non-protein organic compound that is loosely attached, participating in enzymatic reactions as a dissociable carrier of chemical groups or electrons; a prosthetic group is defined as a tightly bound, nonpolypeptide unit in a protein that is regenerated in each enzymatic turnover.)
Some enzymes or enzyme complexes require several cofactors. For example, the multienzyme complex pyruvate dehydrogenase at the junction of glycolysis and the citric acid cycle requires five organic cofactors and one metal ion: loosely bound thiamine pyrophosphate (TPP), covalently bound lipoamide and flavin adenine dinucleotide (FAD), cosubstrates nicotinamide adenine dinucleotide (NAD+) and coenzyme A (CoA), and a metal ion (Mg2+).
Organic cofactors are often vitamins or made from vitamins. Many contain the nucleotide adenosine monophosphate (AMP) as part of their structures, such as ATP, coenzyme A, FAD, and NAD+. This common structure may reflect a common evolutionary origin as part of ribozymes in an ancient RNA world. It has been suggested that the AMP part of the molecule can be considered to be a kind of "handle" by which the enzyme can "grasp" the coenzyme to switch it between different catalytic centers.
Classification.
Cofactors can be divided into two major groups: organic cofactors, such as flavin or heme; and inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+ and iron-sulfur clusters.
Organic cofactors are sometimes further divided into "coenzymes" and "prosthetic groups". The term coenzyme refers specifically to enzymes and, as such, to the functional properties of a protein. On the other hand, "prosthetic group" emphasizes the nature of the binding of a cofactor to a protein (tight or covalent) and, thus, refers to a structural property. Different sources give slightly different definitions of coenzymes, cofactors, and prosthetic groups. Some consider tightly bound organic molecules as prosthetic groups and not as coenzymes, while others define all non-protein organic molecules needed for enzyme activity as coenzymes, and classify those that are tightly bound as coenzyme prosthetic groups. These terms are often used loosely.
A 1980 letter in "Trends in Biochemistry Sciences" noted the confusion in the literature and the essentially arbitrary distinction made between prosthetic groups and coenzymes group and proposed the following scheme. Here, cofactors were defined as an additional substance apart from protein and substrate that is required for enzyme activity and a prosthetic group as a substance that undergoes its whole catalytic cycle attached to a single enzyme molecule. However, the author could not arrive at a single all-encompassing definition of a "coenzyme" and proposed that this term be dropped from use in the literature.
Inorganic cofactors.
Metal ions.
Metal ions are common cofactors. The study of these cofactors falls under the area of bioinorganic chemistry. In nutrition, the list of essential trace elements reflects their role as cofactors. In humans this list commonly includes iron, magnesium, manganese, cobalt, copper, zinc, and molybdenum. Although chromium deficiency causes impaired glucose tolerance, no human enzyme that uses this metal as a cofactor has been identified. Iodine is also an essential trace element, but this element is used as part of the structure of thyroid hormones rather than as an enzyme cofactor. Calcium is another special case, in that it is required as a component of the human diet, and it is needed for the full activity of many enzymes, such as nitric oxide synthase, protein phosphatases, and adenylate kinase, but calcium activates these enzymes in allosteric regulation, often binding to these enzymes in a complex with calmodulin. Calcium is, therefore, a cell signaling molecule, and not usually considered a cofactor of the enzymes it regulates.
Other organisms require additional metals as enzyme cofactors, such as vanadium in the nitrogenase of the nitrogen-fixing bacteria of the genus "Azotobacter", tungsten in the aldehyde ferredoxin oxidoreductase of the thermophilic archaean "Pyrococcus furiosus", and even cadmium in the carbonic anhydrase from the marine diatom "Thalassiosira weissflogii".
In many cases, the cofactor includes both an inorganic and organic component. One diverse set of examples is the heme proteins, which consist of a porphyrin ring coordinated to iron.
Iron–sulfur clusters.
Iron–sulfur clusters are complexes of iron and sulfur atoms held within proteins by cysteinyl residues. They play both structural and functional roles, including electron transfer, redox sensing, and as structural modules.
Organic.
Organic cofactors are small organic molecules (typically a molecular mass less than 1000 Da) that can be either loosely or tightly bound to the enzyme and directly participate in the reaction. In the latter case, when it is difficult to remove without denaturing the enzyme, it can be called a prosthetic group. It is important to emphasize that there is no sharp division between loosely and tightly bound cofactors. Indeed, many such as NAD+ can be tightly bound in some enzymes, while it is loosely bound in others. Another example is thiamine pyrophosphate (TPP), which is tightly bound in transketolase or pyruvate decarboxylase, while it is less tightly bound in pyruvate dehydrogenase. Other coenzymes, flavin adenine dinucleotide (FAD), biotin, and lipoamide, for instance, are tightly bound. Tightly bound cofactors are, in general, regenerated during the same reaction cycle, while loosely bound cofactors can be regenerated in a subsequent reaction catalyzed by a different enzyme. In the latter case, the cofactor can also be considered a substrate or cosubstrate.
Vitamins can serve as precursors to many organic cofactors (e.g., vitamins B1, B2, B6, B12, niacin, folic acid) or as coenzymes themselves (e.g., vitamin C). However, vitamins do have other functions in the body. Many organic cofactors also contain a nucleotide, such as the electron carriers NAD and FAD, and coenzyme A, which carries acyl groups. Most of these cofactors are found in a huge variety of species, and some are universal to all forms of life. An exception to this wide distribution is a group of unique cofactors that evolved in methanogens, which are restricted to this group of archaea.
Cofactors as metabolic intermediates.
Metabolism involves a vast array of chemical reactions, but most fall under a few basic types of reactions that involve the transfer of functional groups. This common chemistry allows cells to use a small set of metabolic intermediates to carry chemical groups between different reactions. These group-transfer intermediates are the loosely bound organic cofactors, often called "coenzymes".
Each class of group-transfer reaction is carried out by a particular cofactor, which is the substrate for a set of enzymes that produce it, and a set of enzymes that consume it. An example of this are the dehydrogenases that use nicotinamide adenine dinucleotide (NAD+) as a cofactor. Here, hundreds of separate types of enzymes remove electrons from their substrates and reduce NAD+ to NADH. This reduced cofactor is then a substrate for any of the reductases in the cell that require electrons to reduce their substrates.
Therefore, these cofactors are continuously recycled as part of metabolism. As an example, the total quantity of ATP in the human body is about 0.1 mole. This ATP is constantly being broken down into ADP, and then converted back into ATP. Thus, at any given time, the total amount of ATP + ADP remains fairly constant. The energy used by human cells requires the hydrolysis of 100 to 150 moles of ATP daily, which is around 50 to 75 kg. In typical situations, humans use up their body weight of ATP over the course of the day. This means that each ATP molecule is recycled 1000 to 1500 times daily.
Evolution.
Organic cofactors, such as ATP and NADH, are present in all known forms of life and form a core part of metabolism. Such universal conservation indicates that these molecules evolved very early in the development of living things. At least some of the current set of cofactors may, therefore, have been present in the last universal ancestor, which lived about 4 billion years ago.
Organic cofactors may have been present even earlier in the history of life on Earth. The nucleotide adenosine is present in cofactors that catalyse many basic metabolic reactions such as methyl, acyl, and phosphoryl group transfer, as well as redox reactions. This ubiquitous chemical scaffold has, therefore, been proposed to be a remnant of the RNA world, with early ribozymes evolving to bind a restricted set of nucleotides and related compounds. Adenosine-based cofactors are thought to have acted as interchangeable adaptors that allowed enzymes and ribozymes to bind new cofactors through small modifications in existing adenosine-binding domains, which had originally evolved to bind a different cofactor. This process of adapting a pre-evolved structure for a novel use is known as "exaptation".
A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH.
History.
The first organic cofactor to be discovered was NAD+, which was identified by Arthur Harden and William Young 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. Other cofactors were identified throughout the early 20th century, with ATP being isolated in 1929 by Karl Lohmann, and coenzyme A being discovered in 1945 by Fritz Albert Lipmann.
The functions of these molecules were at first mysterious, but, in 1936, Otto Heinrich Warburg identified the function of NAD+ in hydride transfer. This discovery was followed in the early 1940s by the work of Herman Kalckar, who established the link between the oxidation of sugars and the generation of ATP. This confirmed the central role of ATP in energy transfer that had been proposed by Fritz Albert Lipmann in 1941. Later, in 1949, Morris Friedkin and Albert L. Lehninger proved that NAD+ linked metabolic pathways such as the citric acid cycle and the synthesis of ATP.
Protein-derived cofactors.
In a number of enzymes, the moiety that acts as a cofactor is formed by post-translational modification of a part of the protein sequence. This often replaces the need for an external binding factor, such as a metal ion, for protein function. Potential modifications could be oxidation of aromatic residues, binding between residues, cleavage or ring-forming. These alterations are distinct from other post-translation protein modifications, such as phosphorylation, methylation, or glycosylation in that the amino acids typically acquire new functions. This increases the functionality of the protein; unmodified amino acids are typically limited to acid-base reactions, and the alteration of resides can give the protein electrophilic sites or the ability to stabilize free radicals. Examples of cofactor production include tryptophan tryptophylquinone (TTQ), derived from two tryptophan side chains, and 4-methylidene-imidazole-5-one (MIO), derived from an Ala-Ser-Gly motif. Characterization of protein-derived cofactors is conducted using X-ray crystallography and mass spectroscopy; structural data is necessary because sequencing does not readily identify the altered sites.
Non-enzymatic cofactors.
The term is used in other areas of biology to refer more broadly to non-protein (or even protein) molecules that either activate, inhibit, or are required for the protein to function. For example, ligands such as hormones that bind to and activate receptor proteins are termed cofactors or coactivators, whereas molecules that inhibit receptor proteins are termed corepressors. One such example is the G protein-coupled receptor family of receptors, which are frequently found in sensory neurons. Ligand binding to the receptors activates the G protein, which then activates an enzyme to activate the effector. In order to avoid confusion, it has been suggested that such proteins that have ligand-binding mediated activation or repression be referred to as coregulators.


Deoxysarpagine hydroxylase

In enzymology, a deoxysarpagine hydroxylase () is an enzyme that catalyzes the chemical reaction
The 4 substrates of this enzyme are 10-deoxysarpagine, NADPH, H+, and O2, whereas its 3 products are sarpagine, NADP+, and H2O.
This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with NADH or NADPH as one donor, and incorporation of one atom o oxygen into the other donor. The systematic name of this enzyme class is 10-deoxysarpagine,NADPH:oxygen oxidoreductase (10-hydroxylating). This enzyme is also called DOSH.


SIGLEC8

Sialic acid-binding Ig-like lectin 8 is a protein that in humans is encoded by the "SIGLEC8" gene. This gene is located on chromosome 19q13.4, about 330 kb downstream of the SIGLEC9 gene. Within the siglec family of transmembrane proteins, Siglec-8 belongs to the CD33-related siglec subfamily, a subfamily that has undergone rapid evolution.
Initial characterization.
Siglec-8 was first identified by CD33 homology screening of ESTs from a cDNA library generated from a patient diagnosed with idiopathic hypereosinophilic syndrome and was originally termed SAF-2 (sialoadhesin family 2).
At the tissue level, Siglec-8 mRNA was found to be most highly expressed in lung, PBMCs, spleen, and kidney.
Expression.
Siglec-8 is expressed by human eosinophils, mast cells, and, to a lesser extent, basophils. It has thus garnered attention as a molecule that is uniquely expressed by immune effector cells involved in asthma and allergy. In both eosinophils and mast cells, Siglec-8 is expressed late in development. Siglec-8 transcript and protein are detectable at day 12 during the in vitro differentiation of eosinophils from cord blood precursors, whereas the transcription factor GATA-1 peaks at day 2 and the secondary granule protein MBP-1 peaks at day 4 in this differentiation system. In mast cells generated from CD34+ precursors, Siglec-8 expression peaks at 4 weeks of differentiation, in parallel with FcεRIα surface expression.
Consistent with the concept that Siglec-8 is a late differentiation marker, Siglec-8 has not been detected on the surface of relatively undifferentiated eosinophilic cell lines, such as EoL-1, AML14, AML14.3D10, or K562, the basophilic leukemia cell line KU812, nor on cells such as HL60 or EoL-3 that have been differentiated towards an eosinophil-like lineage. Only low levels are detected on the human mast cell sub-line HMC-1.1; however, the HMC-1.2 cell line, which bears a second KIT mutation (D816V, in addition to the V560G mutation found in both HMC-1.1 and HMC-1.2 cells) that may induce further differentiation, expresses Siglec-8 at the cell surface. However, based on a small sampling of patients, all eosinophils from patients with chronic eosinophilic leukemia (CEL), hypereosinophilic syndrome, or chronic myeloid leukemia (CML), all basophils from patients with CEL or CML, and all bone marrow mast cells from patients with indolent systemic mastocytosis or aplastic anemia express Siglec-8, providing a potential target for these cells in the context of these hematologic malignancies.
In addition, baboon eosinophils as well as monocytes, a subset of lymphocytes, and neutrophils express on their cell surface a protein or proteins that are recognized by polyclonal human Siglec-8-specific antibody, consistent with genetic analyses indicating the existence of a Siglec-8 ortholog in this species. However, the 2C4, 2E2, and 7C9 monoclonal antibodies against human Siglec-8 were not found to bind to targets on baboon cells, indicating that these particular epitopes are not conserved.
Structure.
Two splice variants of Siglec-8 exist. The initially characterized form contains 431 amino acid residues in total, 47 of which comprise an uncharacteristically short cytoplasmic tail compared to most CD33-associated siglecs. Subsequently, a longer form of Siglec-8, initially termed Siglec-8L, that contains 499 amino acid residues was identified. This longer form of Siglec-8 shares the same extracellular region but includes a longer cytoplasmic tail with two tyrosine-based motifs (an immunoreceptor tyrosine-based inhibitory motif [ITIM] and an immunoreceptor tyrosine-based switch motif [ITSM]). Both forms of Siglec-8 are found in eosinophils and contain a V-set domain with lectin activity and two C2-type Ig repeat domains in the extracellular region. Given that the longer version is felt to be the normal version, the term Siglec-8 is best used to refer to the 499 amino acid version, while the 431 amino acid version is best referred to as the “short form” of Siglec-8.
Ligand binding.
Potential glycan ligands for Siglec-8 have been screened by glycan array. The glycan NeuAcα2–3(6-O-sulfo)Galβ1–4[Fucα1–3]GlcNAc, also known as 6′-sulfo-sialyl Lewis X, binds with high affinity to both Siglec-8 and to a mouse siglec, Siglec-F, which appears to have acquired a similar but not identical function and pattern of expression to human Siglec-8 through convergent evolution (the two siglecs are not orthologous). Rescreening on a more expanded glycan array reconfirmed this finding, but also identified a second closely related ligand in which the fucose is absent (NeuAcα2–3(6-O-sulfo)Galβ1–4GlcNAc, or 6′-sulfated sialyl N-acetyl-D-lactosamine. These interactions are quite specific; no binding could be detected between these siglecs and unsulfated sialyl Lewis X or sialyl Lewis X sulfated at carbon 6 of GlcNAc (6-sulfo-sialyl Lewis X) rather than carbon 6 of galactose as in 6′-sulfo-sialyl Lewis X. Similarly, no other siglecs bind effectively to these Siglec-8 ligands, as demonstrated by selective binding to eosinophils in human blood of a polymer decorated with 6′-sulfo-sialyl Lewis X. The natural ligand or ligands for Siglec-8 have not yet been positively identified, but ongoing studies have determined that there are sialidase-sensitive glycoprotein ligands for Siglec-F in mouse airways that require the activity of the α2,3 sialyltransferase 3 (ST3Gal-III) enzyme for their generation.
Signaling and function.
Eosinophils.
Consistent with the role of most siglecs and the presence of the intracellular ITIM, Siglec-8 has been found to function as an inhibitory immunoregulatory receptor. Ligation of Siglec-8 induces apoptosis in eosinophils, and, surprisingly, the normally pro-survival cytokines interleukin (IL)-5 and GM-CSF have been found to potentiate this apoptotic effect. IL-33, which activates and maintains eosinophils, also exerts a similar potentiating effect on Siglec-8-induced apoptosis. Inhibitor studies demonstrate that apoptosis induced by crosslinking Siglec-8 through the use of an anti-Siglec-8 mAb and a secondary antibody is mediated sequentially through reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and caspase activation. In the presence of IL-5, the loss of mitochondrial membrane integrity is accelerated and the secondary crosslinking antibody is no longer necessary to induce apoptosis. IL-5 stimulation also appears to alter the mode of cell death of eosinophils induced by Siglec-8 ligation in that cell death becomes a caspase-independent process. Costimulation of the IL-5 receptor and Siglec-8 leads to a type of cell death resembling regulated necrosis that is promoted by MEK1/ERK signaling. Because inhibition of MEK1 does not alter ROS generation but the ROS inhibitor diphenyleneiodonium inhibits ERK1/2 phosphorylation and cell death, the production of ROS appears to be upstream of MEK1/ERK signaling in this pathway. Cell death induced by Siglec-8 in the presence of IL-33, in contrast, is mediated primarily by a caspase-dependent pathway, and IL-33 is capable of synergizing with IL-5 in potentiating cell death induced by Siglec-8 ligation.
Mast cells and basophils.
While Siglec-8 ligation does not cause mast cell apoptosis, it inhibits FcεRIα-mediated Ca2+ flux and release of prostaglandin D2 and histamine. However, the release of IL-8 is not prevented by Siglec-8 ligation in mast cells. In experiments using the rat basophilic leukemia cell line RBL-2H3 stably transfected with Siglec-8, the inhibitory effect of Siglec-8 ligation on FcεRIα-mediated degranulation and Ca2+ flux was found to be dependent on the intact ITIM. There are no published data regarding the function of Siglec-8 on basophils.
Relationships with other siglecs.
CD33-related siglec subfamily.
Due to its high level of sequence homology with CD33 (Siglec-3), Siglec-8 is grouped within the CD33-related siglec subfamily. This family is composed of a rapidly evolving group of siglecs that share 50–99% sequence identity. Most members of the subfamily also possess conserved cytoplasmic ITIM and ITIM-like sequences.
Mouse Siglec-F.
While "SIGLEC8" and mouse "Siglecf " do not appear to derive from the same ancestral gene (they are paralogous, not orthologous), they share a binding preference for 6′-sulfo-sialyl Lewis X and 6′-sulfated sialyl "N"-acetyl-D-lactosamine, similar but distinct patterns of cellular expression, and similar inhibitory functions. For example, Siglec-F is expressed by eosinophils, like Siglec-8, but is also expressed by alveolar macrophages and has not been detected on mouse mast cells or basophils. This functional convergence of Siglec-8 and Siglec-F has permitted in vivo studies to be performed in mouse models of eosinophil-mediated disorders that may provide information about the human system. In a chicken ovalbumin (OVA) model of allergic airway inflammation, the Siglec-F knockout mouse exhibits increased lung eosinophilia, enhanced inflammation, delayed resolution, and exacerbated peribronchial fibrosis. Antibody ligation of Siglec-F has also been shown to inhibit eosinophil-mediated intestinal inflammation and airway remodeling in OVA challenge models. The ST3Gal-III enzyme is necessary for the generation of the natural Siglec-F ligand, which remains unknown but is induced by IL-4 and IL-13 in the airway. Loss of this enzyme leads to enhanced allergic eosinophilic airway inflammation. Despite evidence that Siglec-F binds specifically to 6′-sulfo-sialyl Lewis X and 6′-sulfated sialyl "N"-acetyl-D-lactosamine, in which galactose is sulfated at carbon 6, mice deficient in the two known galactose 6-"O"-sulfotransferases, keratan sulfate galactose 6-"O"-sulfotransferase (KSGal6ST) and chondroitin 6-"O"-sulfotransferase 1 (C6ST-1), express equivalent levels of Siglec-F ligand. These models may shed some light on the regulation of human eosinophil biology by Siglec-8 and the production of natural Siglec-8 ligands in humans. Also like Siglec-8, Siglec-F ligation leads to the apoptosis of eosinophils. However, Siglec-F–induced eosinophil apoptosis is mediated by a mechanism distinct from that employed by Siglec-8, hindering direct comparisons between the mouse and human systems. Siglec-F-induced apoptosis is mediated by caspase activation in mouse eosinophils and does not involve ROS, in contrast to the mechanism reported in Siglec-8–induced apoptosis of human eosinophils. This apoptotic mechanism also does not involve Src family kinases, SHP-1, or NADPH.
Research.
In a randomized clinical trial, lirentelimab, a monoclonal antibody targeting SIGLEC8 has been evaluated as a treatment for eosinophilic gastritis and duodenitis.


Piceatannol

Piceatannol is a stilbenoid, a type of phenolic compound.
Natural occurrences.
Piceatannol and its glucoside, astringin, are phenolic compounds found in mycorrhizal and non-mycorrhizal roots of Norway spruces ("Picea abies"). It can also be found in the seeds of the palm "Aiphanes horrida" and in "Gnetum cleistostachyum". The chemical structure of piceatannol was established by Cunningham et al. as being an analog of resveratrol.
In food.
Piceatannol is a metabolite of resveratrol found in red wine, grapes, passion fruit, white tea, and Japanese knotweed. Astringin, a piceatannol glucoside, is also found in red wine.
Biochemical study.
A 1989 "in vitro" study found that piceatannol blocked LMP2A, a viral protein-tyrosine kinase implicated in leukemia, non-Hodgkin's lymphoma and other diseases associated with Epstein-Barr virus. In 2003, this prompted research interest in piceatannol and its effect on these diseases.
Injected in rats, piceatannol shows a rapid glucuronidation and a poor bioavailability, according to a 2006 study.
A 2012 Purdue University study found that fat cells in culture, in the presence of piceatannol, alters the timing of gene expressions, gene functions and insulin action, resulting in the delay or complete inhibition of adipogenesis.


Alcohol dehydrogenase (acceptor)

In enzymology, an alcohol dehydrogenase (acceptor) () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are primary alcohol and acceptor, whereas its two products are aldehyde and reduced acceptor.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with other acceptors. The systematic name of this enzyme class is alcohol:acceptor oxidoreductase. Other names in common use include primary alcohol dehydrogenase, MDH, quinohemoprotein alcohol dehydrogenase, quinoprotein alcohol dehydrogenase, quinoprotein ethanol dehydrogenase, and alcohol:(acceptor) oxidoreductase. This enzyme participates in 5 metabolic pathways: glycolysis / gluconeogenesis, 1,2-dichloroethane degradation, propanoate metabolism, butanoate metabolism, and methane metabolism. It employs one cofactor, PQQ.
Structural studies.
As of late 2007, 11 structures have been solved for this class of enzymes, with PDB accession codes , , , , , , , , , , and .


Tilmacoxib

Tilmacoxib or JTE-522 is a COX-2 inhibitor and is an effective chemopreventive agent against rat experimental liver fibrosis.


Serinolamide A

Serinolamide A is a naturally occurring eicosanoid derivative related to anandamide, which has been isolated from the marine cyanobacteria "Lyngbya majuscula" and related species in the "Oscillatoria" family. Testing established that serinolamide A is an active cannabinoid agonist with moderate potency, having a Ki of 1300 nM at CB1 and five fold selectivity over the related CB2 receptor.


Glycoside hydrolase family 68

In molecular biology, glycoside hydrolase family 68 is a family of glycoside hydrolases.
Glycoside hydrolases are a widespread group of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycoside hydrolases, based on sequence similarity, has led to the definition of >100 different families. This classification is available on the CAZy web site, and also discussed at CAZypedia, an online encyclopedia of carbohydrate active enzymes.
The glycosyl hydrolase 68 family (CAZY GH_68) includes several bacterial levansucrase enzymes, and invertase from "Zymomonas". Levansucrase (), also known as beta-D-fructofuranosyl transferase, catalyses the conversion of sucrose and (2,6-beta-D-fructosyl)(N) to glucose and (2,6-beta-D-fructosyl)(N+1), where other sugars can also act as fructosyl acceptors. Invertase, or extracellular sucrase (), catalyses the hydrolysis of terminal non-reducing beta-D-fructofuranoside residues in beta-D-fructofuranosides.


2-oxoglutarate synthase

In enzymology, a 2-oxoglutarate synthase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are 2-oxoglutarate, CoA, and oxidized ferredoxin, whereas its 3 products are succinyl-CoA, CO2, and reduced ferredoxin.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the aldehyde or oxo group of donor with an iron-sulfur protein as acceptor. The systematic name of this enzyme class is 2-oxoglutarate:ferredoxin oxidoreductase (decarboxylating). Other names in common use include 2-ketoglutarate ferredoxin oxidoreductase, 2-oxoglutarate:ferredoxin oxidoreductase, KGOR, 2-oxoglutarate ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin 2-oxidoreductase (CoA-succinylating). This enzyme participates in the Citric acid cycle. Some forms catalyze the reverse reaction within the Reverse Krebs cycle, as a means of carbon fixation.


Omega loop

The omega loop is a non-regular protein structural motif, consisting of a loop of six or more amino acid residues and any amino acid sequence. The defining characteristic is that residues that make up the beginning and end of the loop are close together in space with no intervening lengths of regular secondary structural motifs. It is named after its shape, which resembles the upper-case Greek letter Omega (Ω).
Structure.
Omega loops, being non-regular, non-repeating secondary structural units, have a variety of three-dimensional shapes. Omega loop shapes are analyzed to identify recurring patterns in dihedral angles and overall loop shape to help identify potential roles in protein folding and function.
Since loops are almost always at the protein surface, it is often assumed that these structures are flexible; however, different omega loops exhibit ranges of flexibility across different time scales of protein motion and have been identified as playing a role in the folding of some proteins, including HIV-1 reverse transcriptase; cytochrome "c"; and nucleases.
Function.
Omega loops can contribute to protein function. For example, omega loops can help stabilize interactions between protein and ligand, such as in the enzyme triose phosphate isomerase, and can directly affect protein function in other enzymes. A heritable coagulation disorder is caused by a single-site mutation in an omega loop of protein C.
Likewise, omega loops play an interesting role in the function of the beta-lactamases: mutations in the "omega loop region" of a beta-lactamase can change its specific function and substrate profile, perhaps due to an important functional role of the correlated dynamics of the region.
Cytochrome "c".
Omega loops have long been recognized also for their importance in the function and folding of the protein cytochrome "c", contributing both key functional residues and well as important dynamic properties. Many researchers have studied omega loop function and dynamics in specific protein systems using a so-called "loop swap" approach, in which loops are swapped between (usually) homologous proteins.


Cerato-platanin

In molecular biology, the cerato-platanin family of proteins includes the phytotoxin cerato-platanin (CP) produced by the Ascomycete "Ceratocystis platani". CP homologs are also found in both the Ascomycota and the Basidiomycota branches of Dikarya. This toxin causes the severe plant disease: canker stain. This protein occurs in the cell wall of the fungus and is involved in the host-pathogen interaction and induces both cell necrosis and phytoalexin synthesis which is one of the first plant defense-related events. CP, like other fungal surface proteins, is able to self-assemble "in vitro". CP is a 120 amino acid protein, containing 40% hydrophobic residues. It is one of the rare examples of protein in which contains a Hopf link. The link is formed by covalent loops - the pieces of protein backbone closed by two disulphide bonds (formed out of four cysteine residues). The N-terminal region of CP is very similar to cerato-ulmin, a phytotoxic protein produced by the "Ophiostoma" species belonging to the hydrophobin family, which also self-assembles.


Mitochondrial thiamine pyrophosphate carrier

Mitochondrial thiamine pyrophosphate carrier is a protein that in humans is encoded by the "SLC25A19" gene.


Diisopropylphosphate

Diisopropylphosphate is an acetylcholinesterase inhibitor. It acts by covalently binding to acetylcholinesterase.


Archease

In molecular biology, the "archease"' superfamily of proteins are represented in all three domains of life. Archease genes are generally located adjacent to genes encoding proteins involved in DNA or RNA processing and therefore been predicted to be modulators or chaperones involved in DNA or RNA metabolism. Many of the roles of archeases remain to be established experimentally.
The function of one of the archeases from the hyperthermophile "Pyrococcus abyssi" has been determined. The gene encoding the archease (PAB1946) is located in a bicistronic operon immediately upstream from a second open reading frame (PAB1947), which encodes a tRNA m5C methyltransferase. The methyl transferase catalyses m5C formation at several cytosine's within tRNAs with preference for C49; the specificity of the methyltransferase reaction being increased by the archease. The archease exists in monomeric and oligomeric states, with only the oligomeric forms able to bind the methyltransferase. Binding prevents aggregation and hinders dimerisation of the methyltransferase-tRNA complex.
The function of this family of archeases as chaperones is supported by structural analysis of the archease from "Methanobacterium thermoautotrophicum", which shows homology to heat shock protein 33, a chaperone protein that inhibits the aggregation of partially denatured proteins.


Diflomotecan

Diflomotecan (also known as BN80905) is a chemotherapeutic agent that is a topoisomerase inhibitor (Top1). It varies from other camptothecin based Top1 inhibitors like topotecan in having a 7-membered E-ring. The oxepan-2-one ring in the homocamptothecin analogues is more stable in plasma compared to the 6-membered lactone in camptothecin. Diflomotecan was the first homocamptothecin to enter clinical trials and is currently in phase I for treating patients with sensitive small cell lung cancer (SCLC).


Bufanolide

Bufanolide is a C24 steroid and, indirectly, a parent structure of bufadienolide. Its derivatives was found in "Bufo" and "Scilla", as an aglycone of cardiac glycosides and is usually toxic.


(S)-norcoclaurine synthase

In enzymology, a ("S")-norcoclaurine synthase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are 4-hydroxyphenylacetaldehyde and 4-(2-aminoethyl)benzene-1,2-diol, whereas its two products are ("S")-norcoclaurine and HO.
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds. The systematic name of this enzyme class is 4-hydroxyphenylacetaldehyde hydro-lyase [adding dopamine ("S")-norcoclaurine-forming]. Other names in common use include ("S")-norlaudanosoline synthase, and 4-hydroxyphenylacetaldehyde hydro-lyase (adding dopamine). This enzyme participates in benzylisoquinoline alkaloid biosynthesis.


List of aldolases

Unqualified, aldolase usually refers to the enzyme fructose-bisphosphate aldolase.
Aldolase may also refer to:


Fucosterol

Fucosterol is a sterol isolated from algae such as "Ecklonia cava" or "Ecklonia stolonifera".


Steroidogenesis inhibitor

A steroidogenesis inhibitor, also known as a steroid biosynthesis inhibitor, is a type of drug which inhibits one or more of the enzymes that are involved in the process of steroidogenesis, the biosynthesis of endogenous steroids and steroid hormones. They may inhibit the production of cholesterol and other sterols, sex steroids such as androgens, estrogens, and progestogens, corticosteroids such as glucocorticoids and mineralocorticoids, and neurosteroids. They are used in the treatment of a variety of medical conditions that depend on endogenous steroids.
Steroidogenesis inhibitors are analogous in effect and use to antigonadotropins (which specifically inhibit sex steroid production), but work via a different mechanism of action; whereas antigonadotropins suppress gonadal production of sex steroids by effecting negative feedback on and thereby suppressing the hypothalamic-pituitary-gonadal axis, steroidogenesis inhibitors directly inhibit the enzymatic biosynthesis of steroids.


Benzoate 4-monooxygenase

In enzymology, a benzoate 4-monooxygenase (, Formerly ) is an enzyme that catalyzes the chemical reaction
The 4 substrates of this enzyme are benzoate, NADPH, H+, and O2, whereas its 3 products are 4-hydroxybenzoate, NADP+, and H2O.
This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with NADH or NADPH as one donor, and incorporation of one atom o oxygen into the other donor. The systematic name of this enzyme class is benzoate,NADPH:oxygen oxidoreductase (4-hydroxylating). Other names in common use include benzoic acid 4-hydroxylase, benzoate 4-hydroxylase, benzoic 4-hydroxylase, benzoate-p-hydroxylase, and p-hydroxybenzoate hydroxylase. This enzyme participates in benzoate degradation via hydroxylation and benzoate degradation via coa ligation. It has 3 cofactors: iron, Tetrahydrobiopterin, and Tetrahydropteridine.


Guanosine nucleotide dissociation inhibitor

In molecular biology, the Guanosine dissociation inhibitors (GDIs) constitute a family of small GTPases that serve a regulatory role in vesicular membrane traffic. GDIs bind to the GDP-bound form of Rho and Rab small GTPases and not only prevent exchange (maintaining the small GTPase in an off-state), but also prevent the small GTPase from localizing at the membrane, which is their place of action. This inhibition can be removed by the action of a GDI displacement factor. GDIs also inhibit cdc42 by binding to its tail and preventing its insertion into membranes; hence it cannot trigger WASPs and cannot lead to nucleation of F-actin.
The GDIs' C-terminal geranylgeranylation is crucial for their membrane association and function. This post-translational modification is catalysed by Rab geranylgeranyl transferase (Rab-GGTase), a multi-subunit enzyme that contains a catalytic heterodimer and an accessory component, termed Rab escort protein (REP)-1. REP-1 presents newly synthesised Rab proteins to the catalytic component, and forms a stable complex with the prenylated proteins following the transfer reaction. The mechanism of REP-1-mediated membrane association of Rab5 is similar to that mediated by Rab GDP dissociation inhibitor (GDI). REP-1 and Rab GDI also share other functional properties, including the ability to inhibit the release of GDP and to remove Rab proteins from membranes.
The crystal structure of the bovine alpha-isoform of Rab GDI has been determined to a resolution of 1.81 Angstrom. The protein is composed of two main structural units: a large complex multi-sheet domain I, and a smaller alpha-helical domain II.
The structural organisation of domain I is closely related to FAD-containing monooxygenases and oxidases. Conserved regions common to GDI and the choroideraemia gene product, which delivers Rab to catalytic subunits of Rab geranylgeranyltransferase II, are clustered on one face of the domain. The two most conserved regions form a compact structure at the apex of the molecule; site-directed mutagenesis has shown these regions to play a critical role in the binding of Rab proteins.


Protein Z

Protein Z (PZ or PROZ) is a protein which in humans is encoded by the "PROZ" gene.
Protein Z is a member of the coagulation cascade, the group of blood proteins that leads to the formation of blood clots. It is a gla domain protein and thus vitamin K-dependent, and its functionality is therefore impaired in warfarin therapy. It is a glycoprotein.
Physiology.
Although it is not enzymatically active, it is structurally related to several serine proteases of the coagulation cascade: factors VII, IX, X and protein C. The carboxyglutamate residues (which require vitamin K) bind protein Z to phospholipid surfaces.
The main role of protein Z appears to be the degradation of factor Xa. This is done by protein Z-related protease inhibitor (ZPI), but the reaction is accelerated 1000-fold by the presence of protein Z. Oddly, ZPI also degrades factor XI, but this reaction does not require the presence of protein Z.
In some studies, deficiency states have been associated with a propensity to thrombosis. Others, however, link it to bleeding tendency; there is no clear explanation for this, as it acts physiologically as an inhibitor, and deficiency would logically have led to a predisposition for thrombosis.
Genetics.
It is 62 kDa large and 396 amino acids long. The "PROZ" gene has been linked to the thirteenth chromosome (13q34).
It has four domains: a gla-rich region, two EGF-like domains and a trypsin-like domain. It lacks the serine residue that would make it catalytically active as a serine protease.
History.
Protein Z was first isolated in cattle blood by Prowse and Esnouf in 1977, and Broze & Miletich determined it in human plasma in 1984.
Structure.
Structural analysis of protein Z will allow better understanding of its function. The Ramachandran plot for protein Z indicates it will form alpha helices. The final structure, all alpha domain, was determined by x-ray diffraction. It consists of chain A and B, which are both helix-loop-helix motifs.


Presenilin

Presenilins are a family of related multi-pass transmembrane proteins which constitute the catalytic subunits of the gamma-secretase intramembrane protease protein complex. They were first identified in screens for mutations causing early onset forms of familial Alzheimer's disease by Peter St George-Hyslop at the Centre for Research in Neurodegenerative Diseases at the University of Toronto, and now also at the University of Cambridge. Vertebrates have two presenilin genes, called "PSEN1" (located on chromosome 14 in humans) that codes for presenilin 1 (PS-1) and "PSEN2" (on chromosome 1 in humans) that codes for presenilin 2 (PS-2). Both genes show conservation between species, with little difference between rat and human presenilins. The nematode worm "C. elegans" has two genes that resemble the presenilins and appear to be functionally similar, sel-12 and hop-1.
Presenilins undergo cleavage in an alpha helical region of one of the cytoplasmic loops to produce a large N-terminal and a smaller C-terminal fragment that together form part of the functional protein. Cleavage of presenilin 1 can be prevented by a mutation that causes the loss of exon 9, and results in loss of function. Presenilins play a key role in the modulation of intracellular Ca2+ involved in presynaptic neurotransmitter release and long-term potentiation induction.
Structure.
Presenilins are transmembrane proteins with nine alpha helices. Structures have been solved of the assembled gamma secretase complex by cryo-electron microscopy, demonstrating significant conformational flexibility in the structure of the presenilin subunit of the complex in response to ligand or inhibitor binding. Presenilins undergo autocatalytic proteolytic processing after expression, cleaving a cytoplasmic loop region between the sixth and seventh helices to produce a large N-terminal and a smaller C-terminal fragment. The two fragments remain in contact with each other in the mature protein. The two catalytic aspartate active site residues required for aspartyl protease activity are located in the sixth and seventh helices.
The structure and membrane topology of presenilins was originally controversial when they were first discovered. The PSEN1 gene was predicted to contain ten trans-membrane domains; models agreed on the expected topology of the N-terminal fragment, but the structure of the C-terminal fragment was disputed. A 2006 study suggested a nine-pass transmembrane topology with cleavage and assembly into the gamma-secretase complex prior to insertion into the plasma membrane. Solution NMR studies of the C-terminal fragment showed three helices likely to traverse the membrane, while X-ray crystallography studies of an archaeal homolog, as well as cryo-electron microscopy of the human gamma-secretase complex, indicate nine transmembrane helices.
Function.
Catalytic.
Presenilins are the catalytic component of the gamma secretase intramembrane protease, a four-member protein complex consisting of presenilin, nicastrin, APH-1, and PEN-2. It has a very broad range of substrates for its proteolytic activity. Its substrates are hydrophobic single-pass transmembrane helices with relatively small extracellular regions. These substrates arise following ectodomain shedding. Well over 100 different integral membrane proteins are processed by gamma-secretase. The best-characterized gamma-secretase substrates are the Notch receptor and amyloid precursor protein (APP). Presenilins' role in the Notch signaling pathway is important in development; mice that have the PS1 gene knocked out die early in development from developmental abnormalities similar to those found when notch is disrupted. In conditional knockout mice where presenilin is only inactivated after early development, there is evidence that presenilins in their role as gamma-secretase components are important in the survival of neurons during aging.
There are subtle and species-specific variations in the roles of presenilin-1 and presenilin-2 in assembled gamma-secretase complexes, with many studies suggesting a primary role for presenilin-1. In humans, the two presenilins differ in subcellular localization, and may also be cell type and tissue-specific.
Non-catalytic.
Presenilins also have additional non-catalytic roles in other cellular signaling processes, including calcium homeostasis, lysosomal acidification, autophagy, and protein trafficking. The proteins' role in calcium homeostasis in neurons has been a subject of interest. The genetic inactivation of presenilins in hippocampal synapses has shown this selectively affects the long-term potentiation caused by theta with the inactivation in presynapse but not the postsynapse impairing short-term plasticity and synaptic facilitation. The release of glutamate was also reduced in presynaptic terminals by processes that involve modulation of intracellular Ca2+ release. This has been suggested to "represent a general convergent mechanism leading to neurodegeneration".
Homologs have been identified and characterized in diverse eukaryotic organisms, including model organisms "Drosophila melanogaster" and "Caenorhabditis elegans", plants such as "Arabidopsis thaliana" and "Physcomitrella patens", and the slime mold "Dictyostelium discoideum". In these functions presenilins are thought to play a role as scaffold proteins, considered likely to be the ancestral role of the protein family.
Expression and distribution.
Both human presenilins have widespread expression in the brain. The two proteins differ in subcellular localization, with PS1 expressed more broadly and present at the cell membrane, while PS2 is present mainly in late endosomes and lysosomes.
Association with Alzheimer's disease.
Most cases of Alzheimer's disease are not hereditary. However, there is a small subset of cases that have an earlier age of onset and have a strong genetic element. In patients suffering from Alzheimer's disease (autosomal dominant hereditary), mutations in the presenilin proteins (PSEN1; PSEN2) or the amyloid precursor protein (APP) can be found. The majority of these cases carry mutant presenilin genes. An important part of the disease process in Alzheimer's disease is the accumulation of Amyloid beta (Aβ) protein. To form Aβ, APP must be cut by two enzymes, beta secretases and gamma secretase. Presenilin is the sub-component of gamma secretase that is responsible for the cutting of APP.
Gamma secretase can cut APP at several points within a small region of the protein, which results in Aβ of various lengths. The lengths associated with Alzheimer's disease are 40 and 42 amino acids long. Aβ 42 is more likely to aggregate to form plaques in the brain than Aβ 40. Presenilin mutations lead to an increase in the ratio of Aβ 42 produced compared to Aβ 40, although the total quantity of Aβ produced remains constant. This can come about by various effects of the mutations upon gamma secretase.
Discovery.
The genes for the presenilins were discovered in 1995 through linkage studies using mutations present in familial Alzheimer's disease cases. Around the same time, the presenilin homolog in "Caenorhabditis elegans", "sel-12", was independently identified as a contributor to Notch signaling. Although the function of the protein products of these genes was not immediately apparent, it became clear from subsequent work that the mutations were associated with higher proportions of Aβ42 over the less amyloidogenic Aβ40. The role of presenilins as the catalytic component of the gamma secretase protein complex was established by the early 2000s.


Gamma-butyrobetaine dioxygenase

Gamma-butyrobetaine dioxygenase (also known as BBOX, GBBH or γ-butyrobetaine hydroxylase) is an enzyme that in humans is encoded by the "BBOX1" gene. Gamma-butyrobetaine dioxygenase catalyses the formation of L-carnitine from gamma-butyrobetaine, the last step in the L-carnitine biosynthesis pathway. Carnitine is essential for the transport of activated fatty acids across the mitochondrial membrane during mitochondrial beta oxidation. In humans, gamma-butyrobetaine dioxygenase can be found in kidney (high), liver (moderate), and brain (very low). "BBOX1" has recently been identified as a potential cancer gene on the basis of a large-scale microarray data analysis.
Reaction.
Gamma-butyrobetaine dioxygenase belongs to the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily. It catalyses the following reaction:
The three substrates of this enzyme are 4-trimethylammoniobutanoate (γ-butyrobetaine), 2-oxoglutarate, and O2, whereas its three products are 3-hydroxy-4-trimethylammoniobutanoate (L-carnitine), succinate, and carbon dioxide.
This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with 2-oxoglutarate as one donor, and incorporation of one atom of oxygen into each donor. This enzyme participates in lysine degradation. Iron is a cofactor for gamma-butyrobetaine dioxygenase. Similar to many other 2OG oxygenases, the activity of gamma-butyrobetaine dioxygenase can be stimulated by reducing agents such as ascorbate and glutathione. The catalytic activity of gamma-butyrobetaine dioxygenase can be stimulated with different metal ions, especially potassium ions.
Both the "apo" (PDB id: 3N6W) and the "holo" (PDB id: 3O2G) structures of gamma-butyrobetaine dioxygenase have been solved, demonstrating an induced fit mechanism may contribute to the catalytic activity of gamma-butyrobetaine dioxygenase.
Gamma-butyrobetaine dioxygenase is promiscuous in substrate selectivity and it processes a number of modified substrates, including the natural catalytic products L-carnitine and D-carnitine, forming 3-dehydrocarnitine and trimethylaminoacetone. Gamma-butyrobetaine dioxygenase also catalyses the oxidation of mildronate to form multiple products including malonic acid semialdehyde, dimethylamine, formaldehyde and (1-methylimidazolidin-4-yl)acetic acid, which is proposed to be formed via a Stevens rearrangement mechanism. Gamma-butyrobetaine dioxygenase is unique among other human 2OG oxygenases that it catalyses both hydroxylation (e.g.: L-carnitine), demethylation (e.g.: formaldehyde) and C-C bond formation (e.g.: (1-methylimidazolidin-4-yl)acetic acid).
Inhibition.
Gamma-butyrobetaine dioxygenase is an inhibition target for 3-(2,2,2-trimethylhydraziniumyl)propionate (mildronate, also known as THP, MET-88, Meldonium or Quarterine). Mildronate is offered, clinically, to non-U.S. markets, in treatment of angina and myocardial infarction. Some studies suggested that mildronate may also be beneficial for the treatment of neurological disorder, diabetes, and seizures and alcohol intoxication. Mildronate is currently manufactured and marketed by Grindeks, a pharmaceutical company based in Latvia. To date, at least five clinical trial reports were published in peer-reviewed journals documenting the efficacy and safety of mildronate on the treatments of angina, stroke and chronic heart failure. However, there have been no randomized clinical trials to support the use of mildronate to treat any cardiovascular disease.
Mildronate has a similar structure to the natural substrate gamma-butyrobetaine, with a NH group replacing the CH2 of gamma-butyrobetaine at the C-4 position. A crystal structure of mldronate in complex with gamma-butyrobetaine dioxygenase was published, and it suggests mildronate bind to gamma-butyrobetaine dioxygenase in exactly the same way as gamma-butyrobetaine (PDB id: 3MS5). To date, most enzyme inhibitors for human 2OG oxygenases bind to the cosubstrate 2OG binding site; mildronate is a rare example of a non-peptidyl substrate mimic inhibitor. Although initial reports suggested mildronate is a non-competitive and non-hydroxylatable analogue of gamma-butyrobetaine, further studies have identified mildronate is indeed a substrate for gamma-butyrobetaine dioxygenase.
Similar to other 2OG oxygenases, gamma-butyrobetaine dioxygenase can be inhibited by 2OG mimics and aromatic inhibitors such as pyridine 2,4-dicarboxylate. Other reported gamma-butyrobetaine dioxygenase inhibitors include cyclopropyl-substituted gamma-butyrobetaines and 3-(2,2-dimethylcyclopropyl)propanoic acid, which is a mechanism-based enzyme inhibitor.
Assay.
Several "in vitro" biochemical assays have been applied to monitor the catalytic activity of gamma-butyrobetaine dioxygenase. Early methods have mainly focused on the use of radiolabeled compounds, including 14C-labelled gamma-butyrobetaine and 14C-labelled 2OG. Enzyme-coupled method have also been applied to detect carnitine formation, by using the enzyme carnitine acetyltransferase and 14C-labelled acetyl-coenzyme A to give labelled acetylcarnitine for detection. Using this method, it is possible to detect carnitine concentration down to the pico-molar range. Other analytical methods including mass spectrometry and NMR have also been applied, and they are in particularly useful for the study of the coupling ratio between 2OG oxidation and substrate formation, and for the characterisation of unknown enzymatic products. However, these methods are often not suitable for high-throughput screening and require expensive instrumentation. A potentially high-throughput fluorescence-based assay has also been proposed by using a fluorinated-gamma-butyrobetaine analog. The fluoride ions released as a result of gamma-butyrobetaine dioxygenase catalyses can be detected by using chemosensors such as protected fluorescein.


Thrombospondin 4

Thrombospondin-4 is a protein that in humans is encoded by the "THBS4" gene.
The protein encoded by this gene belongs to the thrombospondin protein family. Thrombospondin family members are adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. This protein forms a pentamer and can bind to heparin and calcium. Studies of the rat counterpart suggest that this protein may be involved in local signaling in the developing and adult nervous system.


Transmembrane domain

Transmembrane domain usually denotes a transmembrane segment of single alpha helix of a transmembrane protein. More broadly, a transmembrane domain is any membrane-spanning protein domain.
Identification of transmembrane helices.
Transmembrane helices are visible in structures of membrane proteins determined by X-ray diffraction. They may also be predicted on the basis of hydrophobicity scales. Because the interior of the bilayer and the interiors of most proteins of known structure are hydrophobic, it is presumed to be a requirement of the amino acids that span a membrane that they be hydrophobic as well. However, membrane pumps and ion channels also contain numerous charged and polar residues within the generally non-polar transmembrane segments.
Using hydrophobicity analysis to predict transmembrane helices enables a prediction in turn of the "transmembrane topology" of a protein; i.e. prediction of what parts of it protrude into the cell, what parts protrude out, and how many times the protein chain crosses the membrane.
Transmembrane helices can also be identified "in silico" using the bioinformatic tool, TMHMM.


TXNDC5

Thioredoxin domain-containing protein 5 is a protein that in humans is encoded by the "TXNDC5" gene.
Function.
This gene encodes a protein disulfide-isomerase. Its expression is induced by hypoxia and its role may be to protect hypoxic cells from apoptosis. This gene can be co-transcribed with the upstream gene "MUTED".


Epimerase and racemase

Epimerases and racemases are isomerase enzymes that catalyze the inversion of stereochemistry in biological molecules.
Racemases catalyze the stereochemical inversion around the asymmetric carbon atom in a substrate having only one center of asymmetry. Epimerases catalyze the stereochemical inversion of the configuration about an asymmetric carbon atom in a substrate having more than one center of asymmetry, thus interconverting epimers.
Human epimerases include methylmalonyl-CoA epimerase, involved in the metabolic breakdown of the amino acids alanine, isoleucine, methionine and valine, and UDP-glucose 4-epimerase, which is used in the final step of galactose metabolism - catalyzing the reversible conversion of UDP-galactose to UDP-glucose.


Laminin

Laminins are high-molecular weight (~400 to ~900 kDa) proteins of the extracellular matrix. They are a major component of the basal lamina (one of the layers of the basement membrane), a protein network foundation for most cells and organs. The laminins are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, and adhesion.
Laminins are heterotrimeric proteins that contain an α-chain, a β-chain, and a γ-chain, found in five, four, and three genetic variants, respectively. The laminin molecules are named according to their chain composition. Thus, laminin-511 contains α5, β1, and γ1 chains. Fourteen other chain combinations have been identified in vivo. The trimeric proteins intersect to form a cross-like structure that can bind to other cell membrane and extracellular matrix molecules. The three shorter arms are particularly good at binding to other laminin molecules, which allows them to form sheets. The long arm is capable of binding to cells, which helps anchor organized tissue cells to the membrane.
The laminin family of glycoproteins are an integral part of the structural scaffolding in almost every tissue of an organism. They are secreted and incorporated into cell-associated extracellular matrices. Laminin is vital for the maintenance and survival of tissues. Defective laminins can cause muscles to form improperly, leading to a form of muscular dystrophy, lethal skin blistering disease (junctional epidermolysis bullosa) and defects of the kidney filter (nephrotic syndrome).
Types.
Fifteen laminin trimers have been identified. The laminins are combinations of different alpha-, beta-, and gamma-chains.
Laminins were previously numbered as they were discovered, i.e. laminin-1, laminin-2, laminin-3, etc., but the nomenclature was changed to describe which chains are present in each isoform (laminin-111, laminin-211, etc.). In addition, many laminins had common names before either laminin nomenclature was in place.
Function.
Laminins form independent networks and are associated with type IV collagen networks via entactin, fibronectin, and perlecan. They also bind to cell membranes through integrin receptors and other plasma membrane molecules, such as the dystroglycan glycoprotein complex and Lutheran blood group glycoprotein. Through these interactions, laminins critically contribute to cell attachment and differentiation, cell shape and movement, maintenance of tissue phenotype, and promotion of tissue survival. Some of these biological functions of laminin have been associated with specific amino-acid sequences or fragments of laminin. For example, the peptide sequence [GTFALRGDNGDNGQ], which is located on the alpha-chain of laminin, promotes adhesion of endothelial cells.
Laminin alpha4 is distributed in a variety of tissues including peripheral nerves, dorsal root ganglion, skeletal muscle and capillaries; in the neuromuscular junction, it is required for synaptic specialisation. The structure of the laminin-G domain has been predicted to resemble that of pentraxin.
Role in neural development.
Laminin-111 is a major substrate along which nerve axons will grow, both in vivo and in vitro. For example, it lays down a path that developing retinal ganglion cells follow on their way from the retina to the tectum. It is also often used as a substrate in cell culture experiments. The presence of laminin-1 can influence how the growth cone responds to other cues. For example, growth cones are repelled by netrin when grown on laminin-111, but are attracted to netrin when grown on fibronectin. This effect of laminin-111 probably occurs through a lowering of intracellular cyclic AMP.
Role in peripheral nerve repair.
Laminins are enriched at the lesion site after peripheral nerve injury and are secreted by Schwann cells. Neurons of the peripheral nervous system express integrin receptors that attach to laminins and promote neuroregeneration after injury.
Pathology.
Dysfunctional structure of one particular laminin, laminin-211, is the cause of one form of congenital muscular dystrophy. Laminin-211 is composed of an α2, a β1 and a γ1 chains. This laminin's distribution includes the brain and muscle fibers. In muscle, it binds to alpha dystroglycan and integrin alpha7—beta1 via the G domain, and via the other end it binds to the extracellular matrix.
Abnormal laminin-332, which is essential for epithelial cell adhesion to the basement membrane, leads to a condition called junctional epidermolysis bullosa, characterized by generalized blisters, exuberant granulation tissue of skin and mucosa, and pitted teeth.
Malfunctional laminin-521 in the kidney filter causes leakage of protein into the urine and nephrotic syndrome.
Role in cancer.
Some of the laminin isoforms have been implicated in cancer pathophysiology. The majority of transcripts that harbor an internal ribosome entry site (IRES) are involved in cancer development via corresponding proteins. A crucial event in tumor progression referred to as epithelial to mesenchymal transition (EMT) allows carcinoma cells to acquire invasive properties. The translational activation of the extracellular matrix component laminin B1 (LAMB1) during EMT has been recently reported suggesting an IRES-mediated mechanism. In this study, the IRES activity of LamB1 was determined by independent bicistronic reporter assays. Strong evidences exclude an impact of cryptic promoter or splice sites on IRES-driven translation of LamB1. Furthermore, no other LamB1 mRNA species arising from alternative transcription start sites or polyadenylation signals were detected that account for its translational control. Mapping of the LamB1 5'-untranslated region (UTR) revealed the minimal LamB1 IRES motif between -293 and -1 upstream of the start codon. Notably, RNA affinity purification showed that the La protein interacts with the LamB1 IRES. This interaction and its regulation during EMT were confirmed by ribonucleoprotein immunoprecipitation. In addition, La was able to positively modulate LamB1 IRES translation. In summary, these data indicate that the LamB1 IRES is activated by binding to La which leads to translational upregulation during hepatocellular EMT.
Use in cell culture.
Together with other major components of the ECM, such as collagens and fibronectin, laminins have been used to enhance mammalian cell culture, especially in the case of pluripotent stem cells, as well as some primary cell cultures, which can be difficult to propagate on other substrates. Two types of naturally-sourced laminins are commercially available. Laminin-111 extracted from mouse sarcomas is one popular laminin type, as well as laminin mixtures from human placenta, which may primarily correspond to laminin-211, 411 or 511, depending on the provider. The various laminin isoforms are practically impossible to isolate from tissues in pure form due to extensive cross-linking and the need for harsh extraction conditions, such as proteolytic enzymes or low pH, that cause degradation. Therefore, recombinant laminins have been produced since the year 2000. This made it possible to test if laminins could have a significant role "in vitro" as they have in the human body. In 2008, two groups independently showed that mouse embryonic stem cells can be grown for months on top of recombinant laminin-511. Later, Rodin "et al." showed that recombinant laminin-511 can be used to create a totally xeno-free and defined cell culture environment to culture human pluripotent ES cells and human iPS cells.
Laminin domains.
Laminins contain several conserved protein domains.
Laminin I and Laminin II.
Laminins are trimeric molecules; laminin-1 is an alpha1 beta1 gamma1 trimer. It has been suggested that the domains I and II from laminin A, B1 and B2 may come together to form a triple helical coiled-coil structure.
Laminin B.
The laminin B domain (also known as domain IV) is an extracellular module of unknown function. It is found in a number of different proteins that include, heparan sulphate proteoglycan from basement membrane, a laminin-like protein from "Caenorhabditis elegans" and laminin. Laminin IV domain is not found in short laminin chains (alpha4 or beta3).
Laminin EGF-like.
Beside different types of globular domains each laminin subunit contains, in its first half, consecutive repeats of about 60 amino acids in length that include eight conserved cysteines. The tertiary structure of this domain is remotely similar in its N-terminus to that of the EGF-like module. It is also known as a 'LE' or 'laminin-type EGF-like' domain. The number of copies of the laminin EGF-like domain in the different forms of laminins is highly variable; from 3 up to 22 copies have been found. In mouse laminin gamma-1 chain, the seventh LE domain has been shown to be the only one that binds with a high affinity to nidogen. The binding-sites are located on the surface within the loops C1-C3 and C5-C6. Long consecutive arrays of laminin EGF-like domains in laminins form rod-like elements of limited flexibility, which determine the spacing in the formation of laminin networks of basement membranes.
Laminin G.
The laminin globular (G) domain, also known as the LNS (Laminin-alpha, Neurexin and Sex hormone-binding globulin) domain, is on average 177 amino acids in length and can be found in one to six copies in various laminin family members as well as in a large number of other extracellular proteins. For example, all laminin alpha-chains have five laminin G domains, all collagen family proteins have one laminin G domain, the CNTNAP proteins have four laminin G domains, while neurexin 1 and 2 each hold six laminin G domains. On average, approximately one quarter of the proteins that hold laminin G domains is taken up by these laminin G domains themselves. The smallest laminin G domain can be found in one of the collagen proteins (COL24A1; 77 AA) and the largest domain in TSPEAR (219 AA).
The exact function of the Laminin G domains has remained elusive, and a variety of binding functions has been ascribed to different Laminin G modules. For example, the laminin alpha1 and alpha2 chains each have five C-terminal laminin G domains, where only domains LG4 and LG5 contain binding sites for heparin, sulphatides and the cell surface receptor dystroglycan. Laminin G-containing proteins appear to have a wide variety of roles in cell adhesion, signalling, migration, assembly and differentiation.
Laminin N-terminal.
Basement membrane assembly is a cooperative process in which laminins polymerise through their N-terminal domain (LN or domain VI) and anchor to the cell surface through their G domains. Netrins may also associate with this network through heterotypic LN domain interactions. This leads to cell signalling through integrins and dystroglycan (and possibly other receptors) recruited to the adherent laminin. This LN domain-dependent self-assembly is considered to be crucial for the integrity of basement membranes, as highlighted by genetic forms of muscular dystrophy containing the deletion of the LN module from the alpha 2 laminin chain. The laminin N-terminal domain is found in all laminin and netrin subunits except laminin alpha 3A, alpha 4 and gamma 2.
Human proteins containing laminin domains.
Laminin Domain I.
LAMA1; LAMA2; LAMA3; LAMA4; LAMA5;
Laminin Domain II.
LAMA1; LAMA2; LAMA3; LAMA4; LAMA5;
Laminin B (Domain IV).
HSPG2; LAMA1; LAMA2; LAMA3; LAMA5; LAMC1; LAMC2; LAMC3;
Laminin EGF-like (Domains III and V).
AGRIN; ATRN; ATRNL1; CELSR1; CELSR2; CELSR3; CRELD1; HSPG2;
LAMA1; LAMA2; LAMA3; LAMA4; LAMA5; LAMB1; LAMB2; LAMB3;
LAMB4; LAMC1; LAMC2; LAMC3; MEGF10; MEGF12; MEGF6;
MEGF8; MEGF9; NSR1; NTN1; NTN2L; NTN4; NTNG1; NTNG2;
RESDA1; SCARF1; SCARF2; SREC; STAB1; USH2A;
Laminin G domain.
AGRIN; CELSR1; CELSR2; CELSR3; CNTNAP1; CNTNAP2; CNTNAP3; CNTNAP3B; CNTNAP4;
CNTNAP5; COL11A1; COL11A2; COL12A1; COL14A1; COL15A1; COL16A1; COL18A1; COL19A1;
COL20A1; COL21A1; COL22A1; COL24A1; COL27A1; COL5A1; COL5A3; COL9A1; CRB1;
CRB2; CSPG4; EGFLAM; EYS; FAT; FAT2; FAT3; FAT4; GAS6; HSPG2;
LAMA1; LAMA2; LAMA3; LAMA4; LAMA5; NELL1; NELL2; NRXN1; NRXN2; NRXN3;
PROS1; SLIT1; SLIT2; SLIT3; SPEAR; THBS1; THBS2; THBS3; THBS4; USH2A;
Laminin N-terminal (Domain VI).
LAMA1; LAMA2; LAMA3; LAMA5; LAMB1; LAMB2; LAMB3; LAMB4;
LAMC1; LAMC3; NTN1; NTN2L; NTN4; NTNG1; NTNG2; USH2A;


UNC45A

Protein unc-45 homolog A is a protein that in humans is encoded by the "UNC45A" gene.


Tropoelastin


List of protein tandem repeat annotation software

Computational methods use different properties of protein sequences and structures to find, characterize and annotate protein tandem repeats.


ARID domain

In molecular biology, the ARID domain (AT-rich interaction domain; also known as BRIGHT (B-cell Regulator of Ig Heavy chain Transcription) domain)) is a protein domain that binds to DNA. ARID domain-containing proteins are found in fungi, plants and invertebrate and vertebrate metazoans. ARID-encoding genes are involved in a variety of biological processes including embryonic development, cell lineage gene regulation and cell cycle control. Although the specific roles of this domain and of ARID-containing proteins in transcriptional regulation are yet to be elucidated, they include both positive and negative transcriptional regulation and a likely involvement in the modification of chromatin structure. The basic structure of the ARID domain appears to be a series of six alpha-helices separated by beta-strands, loops, or turns, but the structured region may extend to an additional helix at either or both ends of the basic six. Based on primary sequence homology, they can be partitioned into three structural classes: Minimal ARID proteins that consist of a core domain formed by six alpha helices; ARID proteins that supplement the core domain with an N-terminal alpha-helix; and Extended-ARID proteins, which contain the core domain and additional alpha-helices at their N- and C-termini.
The human SWI-SNF complex protein ARID1A is an ARID family member with non-sequence-specific DNA binding activity. The ARID consensus and other structural features are common to both ARID1A and yeast SWI1, suggesting that ARID1A is a human counterpart of SWI1. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a "Drosophila melanogaster" gene product required for normal development), and MRF-2 (which represses expression from the Cytomegalovirus enhancer) have been analyzed directly with regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, ARID1A shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.