Serpin

Serpins are a superfamily of proteins with similar structures that were first identified for their protease inhibition activity and are found in all kingdoms of life. The acronym serpin was originally coined because the first serpins to be identified act on chymotrypsin-like serine proteases (serine protease inhibitors). They are notable for their unusual mechanism of action, in which they irreversibly inhibit their target protease by undergoing a large conformational change to disrupt its active site. This contrasts with the more common competitive mechanism for protease inhibitors that bind to and block access to the protease active site.
Protease inhibition by serpins controls an array of biological processes, including coagulation and inflammation, and consequently these proteins are the target of medical research. Their unique conformational change also makes them of interest to the structural biology and protein folding research communities. The conformational-change mechanism confers certain advantages, but it also has drawbacks: serpins are vulnerable to mutations that can result in serpinopathies such as protein misfolding and the formation of inactive long-chain polymers. Serpin polymerisation not only reduces the amount of active inhibitor, but also leads to accumulation of the polymers, causing cell death and organ failure.
Although most serpins control proteolytic cascades, some proteins with a serpin structure are not enzyme inhibitors, but instead perform diverse functions such as storage (as in egg white—ovalbumin), transport as in hormone carriage proteins (thyroxine-binding globulin, cortisol-binding globulin) and molecular chaperoning (HSP47). The term "serpin" is used to describe these members as well, despite their non-inhibitory function, since they are evolutionarily related.
History.
Protease inhibitory activity in blood plasma was first reported in the late 1800s, but it was not until the 1950s that the serpins antithrombin and alpha 1-antitrypsin were isolated. Initial research focused on their role in human disease: alpha 1-antitrypsin deficiency is one of the most common genetic disorders, causing emphysema, and antithrombin deficiency results in thrombosis.
In the 1980s, it became clear that these inhibitors were part of superfamily of related proteins that included both protease inhibitors (e.g. alpha 1-antitrypsin) and non-inhibitory members (e.g. ovalbumin). The name "serpin" was coined based on the most common activity of the superfamily (serine protease inhibitors). Around the same time, the first structures were solved for serpin proteins (first in the relaxed, and later in the stressed conformation). The structures indicated that the inhibitory mechanism involved an unusual conformational change and prompted the subsequent structural focus of serpin studies.
Over 1000 serpins have now been identified, including 36 human proteins, as well as molecules in all kingdoms of life—animals, plants, fungi, bacteria, and archaea—and some viruses. In the 2000s, a systematic nomenclature was introduced in order to categorise members of the serpin superfamily based on their evolutionary relationships. Serpins are therefore the largest and most diverse superfamily of protease inhibitors.
Activity.
Most serpins are protease inhibitors, targeting extracellular, chymotrypsin-like serine proteases. These proteases possess a nucleophilic serine residue in a catalytic triad in their active site. Examples include thrombin, trypsin, and human neutrophil elastase. Serpins act as irreversible, suicide inhibitors by trapping an intermediate of the protease's catalytic mechanism.
Some serpins inhibit other protease classes, typically cysteine proteases, and are termed "cross-class inhibitors". These enzymes differ from serineproteases in that they use a nucleophilic cysteine residue, rather than a serine, in their active site. Nonetheless, the enzymatic chemistry is similar, and the mechanism of inhibition by serpins is the same for both classes of protease. Examples of cross-class inhibitory serpins include serpin B4 a squamous cell carcinoma antigen 1 (SCCA-1) and the avian serpin myeloid and erythroid nuclear termination stage-specific protein (MENT), which both inhibit papain-like cysteine proteases.
Biological function and localization.
Protease inhibition.
Approximately two-thirds of human serpins perform extracellular roles, inhibiting proteases in the bloodstream in order to modulate their activities. For example, extracellular serpins regulate the proteolytic cascades central to blood clotting (antithrombin), the inflammatory and immune responses (antitrypsin, antichymotrypsin, and C1-inhibitor) and tissue remodelling (PAI-1). By inhibiting signalling cascade proteases, they can also affect development. The table of human serpins (below) provides examples of the range of functions performed by human serpin, as well as some of the diseases that result from serpin deficiency.
The protease targets of intracellular inhibitory serpins have been difficult to identify, since many of these molecules appear to perform overlapping roles. Further, many human serpins lack precise functional equivalents in model organisms such as the mouse. Nevertheless, an important function of intracellular serpins may be to protect against the inappropriate activity of proteases inside the cell. For example, one of the best-characterised human intracellular serpins is Serpin B9, which inhibits the cytotoxic granule protease granzyme B. In doing so, Serpin B9 may protect against inadvertent release of granzyme B and premature or unwanted activation of cell death pathways.
Some viruses use serpins to disrupt protease functions in their host. The cowpox viral serpin CrmA (cytokine response modifier A) is used in order to avoid inflammatory and apoptotic responses of infected host cells. CrmA increases infectivity by suppressing its host's inflammatory response through inhibition of IL-1 and IL-18 processing by the cysteine protease caspase-1. In eukaryotes, a plant serpin inhibits both metacaspases and a papain-like cysteine protease.
Non-inhibitory roles.
Non-inhibitory extracellular serpins also perform a wide array of important roles. Thyroxine-binding globulin and transcortin transport the hormones thyroxine and cortisol, respectively. The non-inhibitory serpin ovalbumin is the most abundant protein in egg white. Its exact function is unknown, but it is thought to be a storage protein for the developing foetus. Heat shock serpin 47 is a chaperone, essential for proper folding of collagen. It acts by stabilising collagen's triple helix whilst it is being processed in the endoplasmic reticulum.
Some serpins are both protease inhibitors and perform additional roles. For example, the nuclear cysteine protease inhibitor MENT, in birds also acts as a chromatin remodelling molecule in a bird's red blood cells.
Structure.
All serpins share a common structure (or fold), despite their varied functions. All typically have three β-sheets (named A, B and C) and eight or nine α-helices (named hA–hI). The most significant regions to serpin function are the A-sheet and the reactive centre loop (RCL). The A-sheet includes two β-strands that are in a parallel orientation with a region between them called the 'shutter', and upper region called the 'breach'. The RCL forms the initial interaction with the target protease in inhibitory molecules. Structures have been solved showing the RCL either fully exposed or partially inserted into the A-sheet, and serpins are thought to be in dynamic equilibrium between these two states. The RCL also only makes temporary interactions with the rest of the structure, and is therefore highly flexible and exposed to the solvent.
The serpin structures that have been determined cover several different conformations, which has been necessary for the understanding of their multiple-step mechanism of action. Structural biology has therefore played a central role in the understanding of serpin function and biology.
Conformational change and inhibitory mechanism.
Inhibitory serpins do not inhibit their target proteases by the typical competitive ("lock-and-key") mechanism used by most small protease inhibitors (e.g. Kunitz-type inhibitors). Instead, serpins use an unusual conformational change, which disrupts the structure of the protease and prevents it from completing catalysis. The conformational change involves the RCL moving to the opposite end of the protein and inserting into β-sheet A, forming an extra antiparallel β-strand. This converts the serpin from a stressed state, to a lower-energy relaxed state (S to R transition).
Serine and cysteine proteases catalyse peptide bond cleavage by a two-step process. Initially, the catalytic residue of the active site triad performs a nucleophilic attack on the peptide bond of the substrate. This releases the new N-terminus and forms a covalent ester-bond between the enzyme and the substrate. This covalent complex between enzyme and substrate is called an acyl-enzyme intermediate. For standard substrates, the ester bond is hydrolysed and the new C-terminus is released to complete catalysis. However, when a serpin is cleaved by a protease, it rapidly undergoes the S to R transition before the acyl-enzyme intermediate is hydrolysed. The efficiency of inhibition depends on fact that the relative kinetic rate of the conformational change is several orders of magnitude faster than hydrolysis by the protease.
Since the RCL is still covalently attached to the protease via the ester bond, the S to R transition pulls protease from the top to the bottom of the serpin and distorts the catalytic triad. The distorted protease can only hydrolyse the acyl enzyme intermediate extremely slowly and so the protease remains covalently attached for days to weeks. Serpins are classed as irreversible inhibitors and as suicide inhibitors since each serpin protein permanently inactivates a single protease, and can only function once.
Allosteric activation.
The conformational mobility of serpins provides a key advantage over static lock-and-key protease inhibitors. In particular, the function of inhibitory serpins can be regulated by allosteric interactions with specific cofactors. The X-ray crystal structures of antithrombin, heparin cofactor II, MENT and murine antichymotrypsin reveal that these serpins adopt a conformation wherein the first two amino acids of the RCL are inserted into the top of the A β-sheet. The partially inserted conformation is important because co-factors are able to conformationally switch certain partially inserted serpins into a fully expelled form. This conformational rearrangement makes the serpin a more effective inhibitor.
The archetypal example of this situation is antithrombin, which circulates in plasma in a partially inserted relatively inactive state. The primary specificity determining residue (the P1 arginine) points toward the body of the serpin and is unavailable to the protease. Upon binding a high-affinity pentasaccharide sequence within long-chain heparin, antithrombin undergoes a conformational change, RCL expulsion, and exposure of the P1 arginine. The heparin pentasaccharide-bound form of antithrombin is, thus, a more effective inhibitor of thrombin and factor Xa. Furthermore, both of these coagulation proteases also contain binding sites (called exosites) for heparin. Heparin, therefore, also acts as a template for binding of both protease and serpin, further dramatically accelerating the interaction between the two parties. After the initial interaction, the final serpin complex is formed and the heparin moiety is released. This interaction is physiologically important. For example, after injury to the blood vessel wall, heparin is exposed, and antithrombin is activated to control the clotting response. Understanding of the molecular basis of this interaction enabled the development of Fondaparinux, a synthetic form of Heparin pentasaccharide used as an anti-clotting drug.
Latent conformation.
Certain serpins spontaneously undergo the S to R transition without having been cleaved by a protease, to form a conformation termed the latent state. Latent serpins are unable to interact with proteases and so are no longer protease inhibitors. The conformational change to latency is not exactly the same as the S to R transition of a cleaved serpin. Since the RCL is still intact, the first strand of the C-sheet has to peel off to allow full RCL insertion.
Regulation of the latency transition can act as a control mechanism in some serpins, such as PAI-1. Although PAI-1 is produced in the inhibitory S conformation, it "auto-inactivates" by changing to the latent state unless it is bound to the cofactor vitronectin. Similarly, antithrombin can also spontaneously convert to the latent state, as an additional modulation mechanism to its allosteric activation by heparin. Finally, the N-terminus of tengpin, a serpin from "Thermoanaerobacter tengcongensis", is required to lock the molecule in the native inhibitory state. Disruption of interactions made by the N-terminal region results in spontaneous conformational change of this serpin to the latent conformation.
Conformational change in non-inhibitory functions.
Certain non-inhibitory serpins also use the serpin conformational change as part of their function. For example, the native (S) form of thyroxine-binding globulin has high affinity for thyroxine, whereas the cleaved (R) form has low affinity. Similarly, transcortin has higher affinity for cortisol when in its native (S) state, than its cleaved (R) state. Thus, in these serpins, RCL cleavage and the S to R transition has been commandeered to allow for ligand release, rather than protease inhibition.
In some serpins, the S to R transition can activate cell signalling events. In these cases, a serpin that has formed a complex with its target protease, is then recognised by a receptor. The binding event then leads to downstream signalling by the receptor. The S to R transition is therefore used to alert cells to the presence of protease activity. This differs from the usual mechanism whereby serpins affect signalling simply by inhibiting proteases involved in a signalling cascade.
Degradation.
When a serpin inhibits a target protease, it forms a permanent complex, which needs to be disposed of. For extracellular serpins, the final serpin-enzyme complexes are rapidly cleared from circulation. One mechanism by which this occurs in mammals is via the low-density lipoprotein receptor-related protein (LRP), which binds to inhibitory complexes made by antithrombin, PA1-1, and neuroserpin, causing cellular uptake. Similarly, the "Drosophila" serpin, necrotic, is degraded in the lysosome after being trafficked into the cell by the Lipophorin Receptor-1 (homologous to the mammalian LDL receptor family).
Disease and serpinopathies.
Serpins are involved in a wide array of physiological functions, and so mutations in genes encoding them can cause a range of diseases. Mutations that change the activity, specificity or aggregation properties of serpins all affect how they function. The majority of serpin-related diseases are the result of serpin polymerisation into aggregates, though several other types of disease-linked mutations also occur. The disorder alpha-1 antitrypsin deficiency is one of the most common hereditary diseases.
Inactivity or absence.
Since the stressed serpin fold is high-energy, mutations can cause them to incorrectly change into their lower-energy conformations (e.g. relaxed or latent) before they have correctly performed their inhibitory role.
Mutations that affect the rate or the extent of RCL insertion into the A-sheet can cause the serpin to undergo its S to R conformational change before having engaged a protease. Since a serpin can only make this conformational change once, the resulting misfired serpin is inactive and unable to properly control its target protease. Similarly, mutations that promote inappropriate transition to the monomeric latent state cause disease by reducing the amount of active inhibitory serpin. For example, the disease-linked antithrombin variants "wibble" and "wobble", both promote formation of the latent state.
The structure of the disease-linked mutant of antichymotrypsin (L55P) revealed another, inactive "δ-conformation". In the δ-conformation, four residues of the RCL are inserted into the top of β-sheet A. The bottom half of the sheet is filled as a result of one of the α-helices (the F-helix) partially switching to a β-strand conformation, completing the β-sheet hydrogen bonding. It is unclear whether other serpins can adopt this conformer, and whether this conformation has a functional role, but it is speculated that the δ-conformation may be adopted by Thyroxine-binding globulin during thyroxine release. The non-inhibitory proteins related to serpins can also cause diseases when mutated. For example, mutations in SERPINF1 cause osteogenesis imperfecta type VI in humans.
In the absence of a required serpin, the protease that it normally would regulate is over-active, leading to pathologies. Consequently, simple deficiency of a serpin (e.g. a null mutation) can result in disease. Gene knockouts, particularly in mice, are used experimentally to determine the normal functions of serpins by the effect of their absence.
Specificity change.
In some rare cases, a single amino acid change in a serpin's RCL alters its specificity to target the wrong protease. For example, the Antitrypsin-Pittsburgh mutation (M358R) causes the α1-antitrypsin serpin to inhibit thrombin, causing a bleeding disorder.
Polymerisation and aggregation.
The majority of serpin diseases are due to protein aggregation and are termed "serpinopathies". Serpins are vulnerable to disease-causing mutations that promote formation of misfolded polymers due to their inherently unstable structures. Well-characterised serpinopathies include α1-antitrypsin deficiency (alpha-1), which may cause familial emphysema, and sometimes liver cirrhosis, certain familial forms of thrombosis related to antithrombin deficiency, types 1 and 2 hereditary angioedema (HAE) related to deficiency of C1-inhibitor, and familial encephalopathy with neuroserpin inclusion bodies (FENIB; a rare type of dementia caused by neuroserpin polymerisation).
Each monomer of the serpin aggregate exists in the inactive, relaxed conformation (with the RCL inserted into the A-sheet). The polymers are therefore hyperstable to temperature and unable to inhibit proteases. Serpinopathies therefore cause pathologies similarly to other proteopathies (e.g. prion diseases) via two main mechanisms. First, the lack of active serpin results in uncontrolled protease activity and tissue destruction. Second, the hyperstable polymers themselves clog up the endoplasmic reticulum of cells that synthesize serpins, eventually resulting in cell death and tissue damage. In the case of antitrypsin deficiency, antitrypsin polymers cause the death of liver cells, sometimes resulting in liver damage and cirrhosis. Within the cell, serpin polymers are slowly removed via degradation in the endoplasmic reticulum. However, the details of how serpin polymers cause cell death remains to be fully understood.
Physiological serpin polymers are thought to form via domain swapping events, where a segment of one serpin protein inserts into another. Domain-swaps occur when mutations or environmental factors interfere with the final stages of serpin folding to the native state, causing high-energy intermediates to misfold. Both dimer and trimer domain-swap structures have been solved. In the dimer (of antithrombin), the RCL and part of the A-sheet incorporates into the A-sheet of another serpin molecule. The domain-swapped trimer (of antitrypsin) forms via the exchange of an entirely different region of the structure, the B-sheet (with each molecule's RCL inserted into its own A-sheet). It has also been proposed that serpins may form domain-swaps by inserting the RCL of one protein into the A-sheet of another (A-sheet polymerisation). These domain-swapped dimer and trimer structures are thought to be the building blocks of the disease-causing polymer aggregates, but the exact mechanism is still unclear.
Therapeutic strategies.
Several therapeutic approaches are in use or under investigation to treat the most common serpinopathy: antitrypsin deficiency. Antitrypsin augmentation therapy is approved for severe antitrypsin deficiency-related emphysema. In this therapy, antitrypsin is purified from the plasma of blood donors and administered intravenously (first marketed as Prolastin). To treat severe antitrypsin deficiency-related disease, lung and liver transplantation has proven effective. In animal models, gene targeting in induced pluripotent stem cells has been successfully used to correct an antitrypsin polymerisation defect and to restore the ability of the mammalian liver to secrete active antitrypsin. Small molecules have also been developed that block antitrypsin polymerisation "in vitro".
Evolution.
Serpins are the most widely distributed and largest superfamily of protease inhibitors. They were initially believed to be restricted to eukaryote organisms, but have since been found in bacteria, archaea and some viruses. It remains unclear whether prokaryote genes are the descendants of an ancestral prokaryotic serpin or the product of horizontal gene transfer from eukaryotes. Most intracellular serpins belong to a single phylogenetic clade, whether they come from plants or animals, indicating that the intracellular and extracellular serpins may have diverged before the plants and animals. Exceptions include the intracellular heat shock serpin HSP47, which is a chaperone essential for proper folding of collagen, and cycles between the cis-Golgi and the endoplasmic reticulum.
Protease-inhibition is thought to be the ancestral function, with non-inhibitory members the results of evolutionary neofunctionalisation of the structure. The S to R conformational change has also been adapted by some binding serpins to regulate affinity for their targets.
Distribution.
Animal.
Human.
The human genome encodes 16 serpin clades, termed serpinA through serpinP, including 29 inhibitory and 7 non-inhibitory serpin proteins. The human serpin naming system is based upon a phylogenetic analysis of approximately 500 serpins from 2001, with proteins named serpinXY, where X is the clade of the protein and Y the number of the protein within that clade. The functions of human serpins have been determined by a combination of biochemical studies, human genetic disorders, and knockout mouse models.
Specialised mammalian serpins.
Many mammalian serpins have been identified that share no obvious orthology with a human serpin counterpart. Examples include numerous rodent serpins (particularly some of the murine intracellular serpins) as well as the uterine serpins. The term uterine serpin refers to members of the serpin A clade that are encoded by the SERPINA14 gene. Uterine serpins are produced by the endometrium of a restricted group of mammals in the Laurasiatheria clade under the influence of progesterone or estrogen. They are probably not functional proteinase inhibitors and may function during pregnancy to inhibit maternal immune responses against the conceptus or to participate in transplacental transport.
Insect.
The "Drosophila melanogaster" genome contains 29 serpin encoding genes. Amino acid sequence analysis has placed 14 of these serpins in serpin clade Q and three in serpin clade K with the remaining twelve classified as orphan serpins not belonging to any clade. The clade classification system is difficult to use for "Drosophila" serpins and instead a nomenclature system has been adopted that is based on the position of serpin genes on the "Drosophila" chromosomes. Thirteen of the "Drosophila" serpins occur as isolated genes in the genome (including Serpin-27A, see below), with the remaining 16 organised into five gene clusters that occur at chromosome positions 28D (2 serpins), 42D (5 serpins), 43A (4 serpins), 77B (3 serpins) and 88E (2 serpins).
Studies on "Drosophila" serpins reveal that Serpin-27A inhibits the Easter protease (the final protease in the Nudel, Gastrulation Defective, Snake and Easter proteolytic cascade) and thus controls dorsoventral patterning. Easter functions to cleave Spätzle (a chemokine-type ligand), which results in toll-mediated signaling. As well as its central role in embryonic patterning, toll signaling is also important for the innate immune response in insects. Accordingly, serpin-27A also functions to control the insect immune response. In "Tenebrio molitor" (a large beetle), a protein (SPN93) comprising two discrete tandem serpin domains functions to regulate the toll proteolytic cascade.
Nematode.
The genome of the nematode worm "C. elegans" contains 9 serpins, all of which lack signal sequences and so are likely intracellular. However, only 5 of these serpins appear to function as protease inhibitors. One, SRP-6, performs a protective function and guards against stress-induced calpain-associated lysosomal disruption. Further, SRP-6 inhibits lysosomal cysteine proteases released after lysosomal rupture. Accordingly, worms lacking SRP-6 are sensitive to stress. Most notably, SRP-6 knockout worms die when placed in water (the hypo-osmotic stress lethal phenotype or Osl). It has therefore been suggested that lysosomes play a general and controllable role in determining cell fate.
Plant.
Plant serpins were amongst the first members of the superfamily that were identified. The serpin barley protein Z is highly abundant in barley grain, and one of the major protein components in beer. The genome of the model plant, "Arabidopsis thaliana" contain 18 serpin-like genes, although only 8 of these are full-length serpin sequences.
Plant serpins are potent inhibitors of mammalian chymotrypsin-like serine proteases "in vitro", the best-studied example being barley serpin Zx (BSZx), which is able to inhibit trypsin and chymotrypsin as well as several blood coagulation factors. However, close relatives of chymotrypsin-like serine proteases are absent in plants. The RCL of several serpins from wheat grain and rye contain poly-Q repeat sequences similar to those present in the prolamin storage proteins of the endosperm. It has therefore been suggested that plant serpins may function to inhibit proteases from insects or microbes that would otherwise digest grain storage proteins. In support of this hypothesis, specific plant serpins have been identified in the phloem sap of pumpkin (CmPS-1) and cucumber plants. Although an inverse correlation between up-regulation of CmPS-1 expression and aphid survival was observed, "in vitro" feeding experiments revealed that recombinant CmPS-1 did not appear to affect insect survival.
Alternative roles and protease targets for plant serpins have been proposed. The "Arabidopsis" serpin, AtSerpin1 (At1g47710; ), mediates set-point control over programmed cell death by targeting the 'Responsive to Desiccation-21' (RD21) papain-like cysteine protease. AtSerpin1 also inhibits metacaspase-like proteases "in vitro". Two other "Arabidopsis" serpins, AtSRP2 (At2g14540) and AtSRP3 (At1g64030) appear to be involved in responses to DNA damage.
Fungal.
A single fungal serpin has been characterized to date: celpin from "Piromyces" spp. strain E2. "Piromyces" is a genus of anaerobic fungi found in the gut of ruminants and is important for digesting plant material. Celpin is predicted to be inhibitory and contains two N-terminal dockerin domains in addition to its serpin domain. Dockerins are commonly found in proteins that localise to the fungal cellulosome, a large extracellular multiprotein complex that breaks down cellulose. It is therefore suggested that celpin may protect the cellulosome against plant proteases. Certain bacterial serpins similarly localize to the cellulosome.
Prokaryotic.
Predicted serpin genes are sporadically distributed in prokaryotes. "In vitro" studies on some of these molecules have revealed that they are able to inhibit proteases, and it is suggested that they function as inhibitors "in vivo". Several prokaryote serpins are found in extremophiles. Accordingly, and in contrast to mammalian serpins, these molecules possess elevated resistance to heat denaturation. The precise role of most bacterial serpins remains obscure, although "Clostridium thermocellum" serpin localises to the cellulosome. It is suggested that the role of cellulosome-associated serpins may be to prevent unwanted protease activity against the cellulosome.
Viral.
Serpins are also expressed by viruses as a way to evade the host's immune defense. In particular, serpins expressed by pox viruses, including cow pox (vaccinia) and rabbit pox (myxoma), are of interest because of their potential use as novel therapeutics for immune and inflammatory disorders as well as transplant therapy. Serp1 suppresses the TLR-mediated innate immune response and allows indefinite cardiac allograft survival in rats. Crma and Serp2 are both cross-class inhibitors and target both serine (granzyme B; albeit weakly) and cysteine proteases (caspase 1 and caspase 8). In comparison to their mammalian counterparts, viral serpins contain significant deletions of elements of secondary structure. Specifically, crmA lacks the D-helix as well as significant portions of the A- and E-helices.


NADH-CoQ reductase

NADH-CoQ reductase may be:


Plakophilin-2

Plakophilin-2 is a protein that in humans is encoded by the "PKP2" gene. Plakophilin 2 is expressed in skin and cardiac muscle, where it functions to link cadherins to intermediate filaments in the cytoskeleton. In cardiac muscle, plakophilin-2 is found in desmosome structures located within intercalated discs. Mutations in "PKP2" have been shown to be causal in arrhythmogenic right ventricular cardiomyopathy.
Structure.
Two splice variants of the "PKP2" gene have been identified. The first has a molecular weight of 97.4 kDa (881 amino acids) and the second of molecular weight of 92.7 kDa (837 amino acids). A processed pseudogene with high similarity to this locus has been mapped to chromosome 12p13.
Plakophilin-2 is a member of the armadillo repeat and plakophilin protein family. Plakophilin proteins contain nine central, conserved armadillo repeat domains flanked by N-terminal and C-terminal domains. Alternately spliced transcripts encoding protein isoforms have been identified.
Plakophilin 2 localizes to cell desmosomes and nuclei and binds plakoglobin, desmoplakin, and the desmosomal cadherins via N-terminal head domain.
Function.
Plakophilin 2 functions to link cadherins to intermediate filaments in the cytoskeleton. In cardiomyocytes, plakophilin-2 is found at desmosome structures within intercalated discs, which link adjacent sarcolemmal membranes together. The desmosomal protein, desmoplakin, is the core constituent of the plaque which anchors intermediate filaments to the sarcolemma by its C-terminus and indirectly to sarcolemmal cadherins by its N-terminus, facilitated by plakoglobin and plakophilin-2. Plakophilin is necessary for normal localization and content of desmoplakin to desmosomes, which may in part be due to the recruitment of protein kinase C alpha to desmoplakin.
Ablation of "PKP2" in mice severely disrupts normal heart morphogenesis. Mutant mice are embryonic lethal and exhibit deficits in the formation of adhering junctions in cardiomyocytes, including the dissociation of desmoplakin and formation of cytoplasmic granular aggregates around embryonic day 10.5-11. Additional malformation included reduced trabeculation, cytoskeletal dissaray and cardiac wall rupture. Further studies demonstrated that plakophilin-2 coordinate with E-cadherin is required to properly localize RhoA early in actin cytoskeletal rearrangement in order to properly couple the assembly of adherens junctions to the translocation of desmosome precursors in newly formed cell-cell junctions.
Plakophilin-2 over time has shown to be more than components of cell-cell junctions; rather the plakophilins are emerging as versatile scaffolds for various signaling pathways that more globally modulate diverse cellular activities. Plakophilin-2 has shown to localize to nuclei, in addition to desmosomal plaques in the cytoplasm. Studies have shown that plakkophillin-2 is found in the nucleoplasm, complexed in the RNA polymerase III holoenzyme with the largest subunit of RNA polymerase III, termed RPC155.
There are data to support molecular crosstalk between plakophilin-2 and proteins involved in mechanical junctions in cardiomyocytes, including connexin 43, the major component of cardiac gap junctions; the voltage-gated sodium channel Na(V)1.5 and its interacting subunit, ankyrin G; and the K(ATP). Decreased expression of plakophilin-2 via siRNA leads to a decrease in and redistribution of connexin 43 protein, as well as a decrease in coupling of adjacent cardiomyocytes. Studies also showed that GJA1 and plakophilin-2 are components in the same biomolecular complex. Plakophilin-2 also associates with Na(V)1.5, and knockdown of plakophilin-2 in cardiomyocytes alters sodium current properties as well as velocity of action potential propagation. It has also been demonstrated that plakophilin-2 associates with an important component of the Na(V)1.5 complex, ankyrin G, and loss of ankyrin G via siRNA downregulation mislocalized plakophilin-2 and connexin 43 in cardiac cells, which was coordinate with decreased electrical coupling of cells and decreased adhesion strength. These studies were further supported by an investigation in a mouse model harboring a "PKP2"-heterozygous null mutation, which showed decreased Na(V)1.5 amplitude, as well as a shift in gating and kinetics; pharmacological challenge also induced ventricular arrhythmias. These findings further support the notion that desmosomes crosstalk with sodium channels in the heart, and suggest that the risk of arrhythmias in patients with "PKP2" mutations may be unveiled with pharmacological challenge. Evidence has also shown that plakophilin-2 binds to the K(ATP) channel subunit, Kir6.2, and that in cardiomyocytes from haploinsufficient "PKP2" mice, K(ATP) channel current density was ∼40% smaller and regional heterogeneity of K(ATP) channels was altered, suggesting that plakophilin-2 interacts with K(ATP) and mediates crosstalk between intercellular junctions and membrane excitability.
Clinical significance.
Mutations in "PKP2" have been associated with, have been shown to cause, and are considered common in arrhythmogenic right ventricular cardiomyopathy, which is characterized by fibrofatty replacement of cardiomyocytes, ventricular tachycardia and sudden cardiac death. It is estimated that 70% of all mutations associated with arrhythmogenic right ventricular cardiomyopathy are within the "PKP2" gene. These mutations in general appear to disrupt the assembly and stability of desmosomes. Mechanistic studies have shown that certain "PKP2" mutations result in instability of the plakophilin-2 protein due to enhanced calpain-mediated degradation.
Specific and sensitive markers of "PKP2" and plakoglobin mutation carriers in arrhythmogenic right ventricular cardiomyopathy have been identified to include T-wave inversions, right ventricular wall motion abnormalities, and ventricular extrasystoles. Additionally, immunohistochemical analysis of proteins comprising cardiomyocyte desmosomes has shown to be a highly sensitive and specific diagnostic indicator.
Clinical and genetic characterization of arrhythmogenic right ventricular cardiomyopathy is currently under intense investigation to understand the penetrance associated with "PKP2" mutations, as well as other genes encoding desmosomal proteins, in disease progression and outcome.
"PKP2" mutations were also found to coexist with sodium channelopathies in patients with Brugada syndrome.
Additionally, plakophilin-2 was found in adherens junctions of cardiac myxomata tumors analyzed, and absent in patients with noncardiac myxomata, suggesting that plakophilin-2 may serve as a valuable marker in the clinical diagnosis of cardiac myxomata.
Interactions.
PKP2 has been shown to interact with:


Alpha,alpha-trehalose-phosphate synthase (UDP-forming)

In enzymology, an alpha,alpha-trehalose-phosphate synthase (UDP-forming) () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are UDP-glucose and D-glucose 6-phosphate, whereas its two products are UDP and alpha,alpha'-trehalose 6-phosphate.
This enzyme belongs to the family of glycosyltransferases, specifically the hexosyltransferases. The systematic name of this enzyme class is "UDP-glucose:D-glucose-6-phosphate 1-alpha-D-glucosyltransferase". Other names in common use include "UDP-glucose-glucose-phosphate glucosyltransferase", "trehalosephosphate-UDP glucosyltransferase", "UDP-glucose-glucose-phosphate glucosyltransferase", "alpha,alpha-trehalose phosphate synthase (UDP-forming)", "phosphotrehalose-uridine diphosphate transglucosylase", "trehalose 6-phosphate synthase", "trehalose 6-phosphate synthetase", "trehalose phosphate synthase", "trehalose phosphate synthetase", "trehalose phosphate-uridine diphosphate glucosyltransferase", "trehalose-P synthetase", "transglucosylase", and "uridine diphosphoglucose phosphate glucosyltransferase". This enzyme participates in starch and sucrose metabolism.
Structural studies.
As of late 2007, 3 structures have been solved for this class of enzymes, with PDB accession codes , , and .


Fatty acid amide

Fatty acid amides (FAAs) are amides formed from a fatty acid and an amine. In nature, many FAAs have ethanolamine as the amine component. Also known as "N"-acylethanolamines, they contain the functionality RC(O)N(H)CH2CH2OH. A well known example is anandamide. Other fatty acid amides are fatty acid primary amides (FAPAs). They contain the functionality RC(O)NH2). Oleamide is an example of this class of FAPAs.
Natural occurrences.
FAAs play a role in intracellular signalling. The signalling is controlled in part by fatty acid amide hydrolases, which convert the amide to the parent fatty acid. One example of signaling is induced by the binding of anandamide to the cannabinoid receptors.
Aliphatic amides can be found in "Zanthoxylum" species found in Nigeria.


Berbamunine synthase

In enzymology, a berbamunine synthase (, Formerly and ) is an enzyme that catalyzes the chemical reaction
The 5 substrates of this enzyme are (S)-N-methylcoclaurine, (R)-N-methylcoclaurine, NADPH, H+, and O2, whereas its 3 products are berbamunine, 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 the other dehydrogenated. The systematic name of this enzyme class is (S)-N-methylcoclaurine,NADPH:oxygen oxidoreductase (C-O phenol-coupling). This enzyme is also called (S)-N-methylcoclaurine oxidase (C-O phenol-coupling). This enzyme participates in alkaloid biosynthesis i.


Prostaglandin F synthase

In enzymology, a prostaglandin-F synthase (PGFS; ) is an enzyme that catalyzes the chemical reaction:
Thus, the two substrates of this enzyme are (5Z,13E)-(15S)-9alpha,11alpha,15-trihydroxyprosta-5,13-dienoate and NADP+, whereas its three products are (5Z,13E)-(15S)-9alpha,15-dihydroxy-11-oxoprosta-5,13-dienoate, NADPH, and H+.
PGFS is a monomeric wild-type protein that was first purified from bovine lung (PDB ID: 2F38). This enzyme belongs to the family of aldo-keto reductase (AKR) based on its high substrate specificity, its high molecular weight (38055.48 Da) and amino acid sequence. In addition, it is categorized as C3 (AKR1C3) because it is an isoform of 3α-hydroxysteroid dehydrogenase.
The function of PGFS is to catalyze the reduction of aldehydes and ketones to their corresponding alcohols. In humans, these reactions take place mostly in the lungs and in the liver. More specifically, PGFS catalyzes the reduction of PGD2 to 9α,11β–PGF2 and PGH2 to PGF2α by using NADPH as cofactor.
Nomenclature.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is (5Z,13E)-(15S)-9alpha,11alpha,15-trihydroxyprosta-5,13-dienoate:NADP+ 11-oxidoreductase.
Other names in common use include prostaglandin-D2 11-reductase, reductase, 15-hydroxy-11-oxoprostaglandin, PGD2 11-ketoreductase, PGF2α synthetase, prostaglandin 11-ketoreductase, prostaglandin D2-ketoreductase, prostaglandin F synthase, prostaglandin F synthetase, synthetase, prostaglandin F2α, prostaglandin-D2 11-reductase, PGF synthetase, NADPH-dependent prostaglandin D2 11-keto reductase, and prostaglandin 11-keto reductase. This enzyme participates in arachidonic acid metabolism.
Structure.
, 7 structures have been solved for this class of enzymes, with PDB accession codes , , , , , , and .
The primary structure of prostaglandin F synthase consists of 323 amino acid residues. The secondary structure consists of 17 α-helices which contain 130 residues and 18 β-strands which contain 55 residues as well as many random coils. The tertiary structure is a single subunit.
The active site of the enzyme is referred to as an (α/β)8 barrel because it consists of 8 α-helices and 8 β-strands. More specifically, the eight α-helices surround the eight β-strands which form the cylindrical core of the active site. In addition, the active site of the enzyme contains also three random coils which help to connect the helices and strands together. The size of the active site of the enzyme is large enough not only to bind NADPH cofactor but also to bind the substrates PGD2 or PGH2.
Reaction.
In order for the PGFS enzyme to catalyze the reduction of the substrates PGH2 or PGD2, the cofactor NADPH must be present in the active site. This cofactor is present deep within the cavity of the enzyme and forms a hydrogen bond with it, whereas the substrate is located closer to the mouth of the cavity which limits its interaction with PGFS. The rate determining step of the catalysis is the binding of NADPH cofactor in the active site of the enzyme. This is because the binding of NADPH occurs before the binding of the substrate. NADPH is an important cofactor because it is involved in the hydride transfer which is necessary for the reduction to take place.
More specifically, in order for the hydride transfer to occur, the substrate (PGD2) has to bind to the active site of the enzyme PGFS. The substrate binds to the active site through hydrogen bonding between the carbonyl group of PGD2 and the hydroxyl group of tyrosine (Y55) as well as one of the imidazole nitrogen of histidine (H117). The hydride shift from NADPH reduces the carbonyl group of PGD2 and forms a new sp3 hydroxyl group (9α,11β–PGF2).
The protonation of the carbonyl oxygen is facilitated at low pH when histidine is used and at high pH when tyrosine is used for hydrogen bonding with the substrate. On the one hand, histidine is an ideal proton donor at low pH because of its pKa value (6.00), which means that it is protonated at a pH below 6.00. On the other hand, tyrosine is an ideal proton donor at higher pH because of its pKa value (10.1). The type of amino acid that is used for protonation depends on the substrate. For example, reduction of PGD2 in the human body occurs at a pH range of 6-9, which makes histidine an ideal proton donor.
The hydride that is transferred to the carbonyl oxygen of PGD2 causes weakening of the hydrogen bond between the substrate and the enzyme. This has as a result the cleavage of the product (9α,11β–PGF2) from the active site of the enzyme.
Use.
In general, prostaglandins are molecules that are used for inflammation, muscle contraction and blood clotting. Prostaglandin F synthase (PGFS) is very important enzyme because it catalyzes the formation of 9α,11β–PGF2 and PGF2α which are critical for the contraction of bronchial, vascular and arterial smooth muscle.
Also, this enzyme can be used in cancer research. Recent studies have shown that there is a correlation between high levels of PGFS in gastrointestinal tumors and the effectiveness of non-steroidal anti-inflammatory drugs (NSAID). The inhibition of PGFS by NSAID could turn out to be a very important medicinal field in the development of anti-cancer medication.
Inhibition.
Prostaglandin F synthase can be inhibited not only by NSAIDs such as indometacin and suprofen but also by a molecule known as bimatoprost (BMP). BMP, an analogue of PGD2 is an ocular hypotensive agent that binds to the active site of the PGFS enzyme. This means that it inhibits the action of PGFS to catalyze the conversion of PGD2 to 9α,11β–PGF2 and PGH2 to PGF2α because it inhibits the substrate to bind to the active site of the enzyme.


Erepsin

Erepsin is a mixture of enzymes contained in a protein fraction found in the intestinal juices that digest peptones into amino acids. It is produced and secreted by the intestinal glands in the ileum and the pancreas, but it is also found widely in other cells. It is, however, a term now rarely used in scientific literature as more precise terms are preferred.
History.
Erepsin was discovered at the beginning of the twentieth century by German physiologist Otto Cohnheim (1873-1953) who found a substance that breaks down peptones into amino acid in the intestines. He termed this hypothetical protease in his protein extract "erepsin" in 1901, derived from a Greek word meaning "I break down" (έρείπω). His discovery was significant as it overturned the previous "hypothesis of resynthesis" which proposed that proteins get broken down into peptones from which proteins may then be resynthesized, and helped establish the idea that free amino acids instead of peptones are the building blocks of protein.
Erepsin was originally thought to be a single enzyme or a mixture of a few enzymes involved in the terminal stages of the breakdown of peptides to free amino acids in the intestines. However, it became clear later that erepsin is in fact a complex mixture of different peptidases. It was also found not to be unique to intestinal mucosa and is present widely in many other cells and organisms. The term erepsin fell from use in scientific literature in the latter half of the twentieth century as scientists considered its use as a term for a single enzyme or a few enzymes misleading, and more precise terms such as aminopeptidase, carboxypeptidase and dipeptidase are preferred. The term is now considered obsolete.
Properties.
Erepsin may contain dipeptidases, aminopeptidases, occasionally carboxypeptidases, and others including leucyl aminopeptidase, prolinase and prolidase. It is often grouped under exopeptidases, proteases that work only on the outermost peptide bonds of a polypeptide chain. The optimum pH for the group of enzymes is around pH 8, but some individual enzymes within this group may be distinguished by their differences in stability and optimum pH.


Isoschizomer

Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other. The first enzyme discovered which recognizes a given sequence is known as the prototype; all subsequently identified enzymes that recognize that sequence are isoschizomers. Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.
In some cases, only one out of a pair of isoschizomers can recognize both the methylated as well as unmethylated forms of restriction sites. In contrast, the other restriction enzyme can recognize only the unmethylated form of the restriction site.
This property of some isoschizomers allows identification of methylation state of the restriction site while isolating it from a bacterial strain.
For example, the restriction enzymes HpaII and MspI are isoschizomers, as they both recognize the sequence 5'-CCGG-3' when it is unmethylated. But when the second C of the sequence is methylated, only MspI can recognize it while HpaII cannot.
An enzyme that recognizes the same sequence but cuts it differently is a neoschizomer. Neoschizomers are a specific type (subset) of isoschizomer. For example, SmaI (CCC/GGG) and XmaI (C/CCGGG) are neoschizomers of each other. Similarly Kpn1 (GGTAC/C) and Acc651 (G/GTACC) are neoschizomers of each other.
An enzyme that recognizes a slightly different sequence, but produces the same ends is an isocaudomer.


Agmatidine

Agmatidine (2-agmatinylcytidine, symbol C+ or agm2C) is a modified cytidine present in the wobble position of the anticodon of several archaeal AUA decoding tRNAs. Agmatidine is essential for correct decoding of the AUA codon in many archaea and is required for aminoacylation of tRNAIle2 with isoleucine.
Introduction.
The genetic code describes how triplet codons on mRNA are translated into protein sequences by specific tRNA molecules which can base-pair with the codons. Precise decoding of the genetic code is a fundamental pre-requisite for long-term survival of all organisms. The nature of the anticodon decides the specificity of hydrogen bonding and hence the accuracy of decoding by tRNAs. Till date, a variety of post-transcriptional modifications have been discovered which aid tRNAs in increasing their repertoire of hydrogen bonding capacities. These modifications usually occur on the first base on the anticodon (position 34 or the wobble base position) which base pairs with the third base on the codon and are critical in specific recognition of codons by tRNAs.
The wobble rules of Crick propose how a limited set of tRNAs can decode a wider set of codons by use of wobble base pairing. These rules have been successful in explaining how most of the genetic code is specifically translated by a limited number of tRNAs. For example, a single phenylalanine tRNA with G in the first anticodon position can base pair with either U or C (thus decoding UUU and UUC) and a single leucine tRNA with a modified U (2-thioU) in the anticodon can base pair with either A or G (thus decoding UUA and UUG).
The mechanism of AUA decoding.
The mechanism of decoding in the box containing AUU, AUC, AUA (all coding for isoleucine) and AUG (coding for methionine) has remained a puzzle for scientists since long. AUU and AUC are decoded by a single isoleucine tRNA (tRNAIle1) which has G in the anticodon while AUA is decoded by a separate tRNA (tRNAIle2). How the second isoleucine tRNA decodes AUA without also decoding AUG has been a subject of much interest over the years.
Different classes of organisms solve the problem of AUA decoding differently. For example, in eukaryotes, a tRNA having inosine at position 34 (IAU anticodon) can decode all three isoleucine codons, while a tRNA having pseudouridine in the anticodon (ψAψ) anticodon can specifically read the AUA codon. In eubacteria, a tRNA having lysidine in the anticodon (LAU) can specifically decode AUA, but not AUG. However, the mechanism by which Archaea solve the problem of AUA decoding was not known till early 2010, when two groups simultaneously published reports that archaeal tRNAIle2 contains a modified cytidine at position 34, which was named agmatidine.
Structure and Biosynthesis.
Agmatidine is similar to lysidine in that the C2-oxo group of cytidine is replaced by the aminoguanidine agmatine instead of by lysine in the case of lysidine. The modification is carried out by the enzyme tRNAIle2 2-agmatinylcytidine synthetase, a product of the gene "tiaS "present in many archaeal members"." Agmatidine is generated in the cell by attachment of agmatine to the C2-oxo group of cytidine by TiaS. Agmatine in turn is a decarboxylation product of arginine (an aminoacid present in all cells).
Agmatidine formation occurs through a three-step mechanism. In step one, TiaS hydrolyzes the α-β phosphodiester bond of ATP to produce AMP and PPi. In step two, the C2 carbonyl oxygen of C34 attacks the γ-phosphorus atom to form the p-C34 intermediate, releasing β-Pi. This is in contrast to the mechanism of lysidine formation where the C2-oxo group is activated by adenylation instead of phosphorylation. In step three, the primary amino group of agmatine attacks the C2 carbon of the p-C34 intermediate to release γ-Pi and form agm2C. TiaS also autophosphorylates its Thr18 with the γ-phosphate of ATP, releasing AMP and β-Pi. This is known to be important for agm2C formation although its exact role is not clear.
Physiology.
Conjugation of agmatine moiety at the C2 carbon of C34 induces a tautomeric conversion of C34 which alters its hydrogen bonding pattern, enabling it to pair with adenosine instead of guanosine. The modification is essential for decoding of AUA codons and a tRNA without the modification is not aminoacylated with isoleucine. Moreover, it has been shown that agmatine is an essential metabolite for the viability of "Thermococcus kodakaraensis."
All of the currently sequenced euryarchaeal and crenarchaeal genomes contain only one annotated isoleucine tRNA and three tRNAs with the CAU anticodon (annotated as methionine tRNAs). Therefore, it is very likely that all members of nanoarchaea and korarchaea use agmatidine modification to selectively read AUA codons. However, currently sequenced genomes from nanoarchaea and korarchaea contain two isoleucone tRNAs, one of which has UAU anticodon (which is probably converted into ψAψ "in-vivo"). Therefore, it is thought that these classes of archaea follow a eukaryote-like strategy to solve the AUA decoding problem.


TAP2

TAP2 is a gene in humans that encodes the protein Antigen peptide transporter 2.
Function.
The membrane-associated protein encoded by this gene is a member of the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the MDR/TAP subfamily. Members of the MDR/TAP subfamily are involved in multidrug resistance. This gene is located 7 kb telomeric to gene family member ABCB2 (TAP1). The protein encoded by this gene is involved in antigen presentation. This protein forms a heterodimer with ABCB2 in order to transport peptides from the cytoplasm to the endoplasmic reticulum. Mutations in this gene may be associated with ankylosing spondylitis, insulin-dependent diabetes mellitus, schizophrenia, and celiac disease. Alternative splicing of this gene produces two products which differ in peptide selectivity and level of restoration of surface expression of MHC class I molecules.


Xylulose

Xylulose is a ketopentose, a monosaccharide containing five carbon atoms, and including a ketone functional group. It has the chemical formula 5105. In nature, it occurs in both the - and -enantiomers. 1-Deoxyxylulose is a precursor to terpenes via the nonmevalonic acid pathway.
Pathology.
-Xylulose accumulates in the urine in patients with pentosuria, due to a deficiency in L-xylulose reductase. Since -xylulose is a reducing sugar like -glucose, pentosuria patients have been wrongly diagnosed in the past to be diabetic.


2-oxoglutarate carboxylase

In enzymology, a 2-oxoglutarate carboxylase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are ATP, 2-oxoglutarate, and HCO3-, whereas its 3 products are ADP, phosphate, and oxalosuccinate.
This enzyme belongs to the family of ligases, specifically those forming carbon-carbon bonds. The systematic name of this enzyme class is . Other names in common use include oxalosuccinate synthetase, carboxylating factor for ICDH (incorrect), CFI, and OGC.


Nudix hydrolase

Nudix hydrolases are a superfamily of hydrolytic enzymes capable of cleaving nucleoside diphosphates linked to x (any moiety), hence their name. The reaction yields nucleoside monophosphate (NMP) plus X-P. Substrates hydrolysed by nudix enzymes comprise a wide range of organic pyrophosphates, including nucleoside di- and triphosphates, dinucleoside and diphosphoinositol polyphosphates, nucleotide sugars and RNA caps, with varying degrees of substrate specificity. Enzymes of the Nudix superfamily are found in all types of organisms, including eukaryotes, bacteria and archaea.
There are two components to the Nudix family: the so-called Nudix fold of a beta sheet with alpha helices on each side and the Nudix motif which contains catalytic and metal-binding amino acids. The Nudix motif is GXXXXXEXXXXXXXREUXEEXGU where U is isoleucine, leucine or valine, and X is any amino acid. This forms a short helix which (usually) contains the catalytic amino acids. Nudix hydrolases include Dcp2 of the decapping complex, ADP-ribose diphosphatase, MutT, ADPRase, Ap4A hydrolases, RppH, and many others.


GABA transaminase inhibitor

In pharmacology, a GABA transaminase inhibitor is an enzyme inhibitor that acts upon GABA transaminase.
Inhibition of GABA transaminase enzymes reduces the degradation of GABA, leading to increased neuronal GABA concentrations.
Examples include valproic acid, vigabatrin, phenylethylidenehydrazine (and drugs that metabolize to it, such as phenelzine), ethanolamine-"O"-sulfate (EOS), and L-cycloserine.
Certain members of this class are used as anticonvulsants.


Cis-trans isomerase

In biochemistry, cis-trans isomerase is a type of isomerase which catalyzes the isomerization of geometric isomers.
Examples include photoisomerase and immunophilins such as cyclophilin.
External links.
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Keratin 20

Keratin 20, often abbreviated CK20, is a protein that in humans is encoded by the "KRT20" gene.
Keratin 20 is a type I cytokeratin. It is a major cellular protein of mature enterocytes and goblet cells and is specifically found in the gastric and intestinal mucosa.
In immunohistochemistry, antibodies to CK20 can be used to identify a range of adenocarcinoma arising from epithelia that normally contain the CK20 protein. For example, the protein is commonly found in colorectal cancer, transitional cell carcinomas and in Merkel cell carcinoma, but is absent in lung cancer, prostate cancer, and non-mucinous ovarian cancer. It is often used in combination with antibodies to CK7 to distinguish different types of glandular tumour.


3-(hydroxyamino)phenol mutase

In enzymology, a 3-(hydroxyamino)phenol mutase () is an enzyme that catalyzes the chemical reaction
Hence, this enzyme has one substrate, 3-hydroxyaminophenol, and one product, aminohydroquinone.
This enzyme belongs to the family of isomerases, specifically those intramolecular transferases transferring hydroxy groups. The systematic name of this enzyme class is 3-(hydroxyamino)phenol hydroxymutase. Other names in common use include 3-hydroxylaminophenol mutase, and 3HAP mutase.


Extra Strong Mints

Extra Strong Mints is a brand name of mints produced in the United Kingdom
History.
The brand was first made in 1935. Trebor was sold to Cadbury in 1989.
Promotion.
In March 1988, the brand gave a £250,000 sponsorship to the England national football team, over three years.
In April 2015, a new series of adverts for the mints (the first in over ten years) was produced by Wieden+Kennedy London.
Production.
They are produced in the north of Sheffield by Trebor, part of Cadbury. Trebor makes around 35 million packs a year at Sheffield.


Aldehyde deformylating oxygenase

Aldehyde deformylating oxygenases (ADO) () are a family of enzymes which catalyze the oxygenation of medium and long chain aldehydes to alkanes via the removal of a carbonyl group as formate.
Aldehyde deformylating oxygenases are found in cyanobacteria as part of the alkane biosynthesis pathway. Their substrates are medium- to long-chain aldehydes formed from acyl-ACP by acyl-ACP reductases (), commonly of 16 and 18 carbons, but potentially as short as nonanal and decanal. Compared to other aldehyde decarbonylases, such as insect or plant aldehyde decarbonylase, cyanobacterial ADO is unusual in evolving formate rather than CO or CO2 and for residing in the cytosol. It is also enzymatically unusual in catalyzing an formally hydrolytic and redox-neutral oxygenation of the substrate.
Structure.
Cyanobacterial aldehyde deformylating oxygenases are cytosolic nonheme di-iron oxygenases, but are much smaller (29 kDa) than other members of the family, and share sequence homology with ferritin-like or ribonucleotide reductases. The overall structure is a bundle of 8 alpha-helices coordinating two central iron cofactors via histidine, aspartate and glutamate. The substrate channels lies parallel to the helices and terminates at the di-iron center.
Conformational changes during the enzymatic cycle of "Synechococcus elongates" ADO have been observed (, , , ). The binding of the substrate aldehyde displaces two coordinating residues on helix 5 (Glu157 and His160), causing a portion of the helix (residues 144-150) to unwind. The resulting hole in the protein surface exposes the active site, facilitating the entrance of the cosubstrate oxygen.
A similar conformational change has been observed for "Prochlorococcus marinus" ADO (), in which residues 154-165 on helix 5 are unwound in the apoenzyme conformation to facilitate metal entry.
Mechanism.
The reaction catalyzed by ADO is unusual in that it is an oxygenation reaction which results in the formal hydrolysis, rather than oxidation, of the substrate. The exact mechanism is not completely understood, and current understanding is based on a consensus between mechanistic studies and comparison with similar enzymes. The structurally similar R2 unit of ribonucleotide reductase proceeds via a tyrosyl radical mechanism, but the homologous tyrosine is replaced by phenylalanine in ADO.
Mechanistic studies suggest that the aldehyde hydrogen is retained in the formate, the alkane hydrogen derives from the solvent, and one formate oxygen originates from O2. The mechanism is tentatively hypothesized to take place by the following steps:
Non-specific formation of alcohols rather than alkanes has also been observed, which would instead correspond to a heterolytic cleavage.
Kinetics.
The Km for O2 is 84 ± 9 µM. However, the observed catalytic turnover is extremely inefficient, on the order of kcat = 1 min−1, raising the possibility that the current understanding of the functional role, cofactors, or even substrates of ADO are incorrect. Transgenetically expressed, ADO appears to be dependent on ferredoxin-ferredoxin reductase to deliver reducing equivalents, but the endogenous reducing system is not known. Further, oxygen-independent aldehyde deformylation has also been observed.
H2O2 is an inhibitor of cADO, and an ADO-catalase fusion protein exhibits improved turnover. Short-chain aldehydes are also observed to be substrate inhibitors.


NLRP7

NACHT, LRR and PYD domains-containing protein 7 is a protein that in humans is encoded by the "NLRP7" gene.
Function.
NALPs are cytoplasmic proteins that form a subfamily within the larger CATERPILLER protein family. Most short NALPs, such as NALP7, have an N-terminal pyrin (MEFV) domain (PYD), followed by a NACHT domain, a NACHT-associated domain (NAD), and a C-terminal leucine-rich repeat (LRR) region. NALPs are implicated in the activation of proinflammatory caspases (e.g., CASP1) via their involvement in multiprotein complexes called inflammasomes.


Histone H2B

Histone H2B is one of the 4 main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and long N-terminal and C-terminal tails, H2B is involved with the structure of the nucleosomes.
Structure.
Histone H2B is a lightweight structural protein made of 126 amino acids. Many of these amino acids have a positive charge at cellular pH, which allows them to interact with the negatively charged phosphate groups in DNA. Along with a central globular domain, histone H2B has two flexible histone tails that extend outwards – one at the N-terminal end and one at C-terminal end. These are highly involved in condensing chromatin from the beads-on-a-string conformation to a 30-nm fiber. Similar to other histone proteins, histone H2B has a distinct histone fold that is optimized for histone-histone as well as histone-DNA interactions.
Two copies of histone H2B come together with two copies each of histone H2A, histone H3, and histone H4 to form the octamer core of the nucleosome to give structure to DNA. To facilitate this formation, histone H2B first binds to histone H2A to form a heterodimer. Two of these heterodimers then bind together with a heterotetramer made of histone H3 and histone H4, giving the nucleosome its characteristic disk shape. DNA is then wrapped around the entire nucleosome in groups of approximately 160 base pairs of DNA. The wrapping continues until all chromatin has been packaged with the nucleosomes.
Function.
Histone H2B is a structural protein that helps organize eukaryotic DNA. It plays an important role in the biology of the nucleus where it is involved in the packaging and maintaining of chromosomes, regulation of transcription, and replication and repair of DNA. Histone H2B helps regulate chromatin structure and function through post-translational modifications and specialized histone variants.
Acetylation and ubiquitination are examples of two post-translational modifications that affect the function of histone H2B in particular ways. Hyperacetylation of histone tails helps DNA-binding proteins access chromatin by weakening histone-DNA and nucleosome-nucleosome interactions. Furthermore, acetylation of a specific lysine residue binds to bromine-containing domains of certain transcription and chromatin regulatory proteins. This docking facilitates the recruitment of these proteins to the correct region of the chromosome. Ubiquitinated histone H2B is often found in regions of active transcription. Through the facilitation of chromatin remodeling, it stimulates transcriptional elongation and sets the stage for further modifications that regulate multiple elements of transcription. Specifically, the ubiquitin on histone H2B opens up and unfolds regions of chromatin allowing transcription machinery access to the promoter and coding regions of DNA.
While only a few isoforms of histone H2B have been studied in depth, researchers have found that histone H2B variants serve important roles. If certain variants stopped functioning, centromeres would not form correctly, genome integrity would be lost, and the DNA damage response would be silenced. Specifically, in some lower eukaryotes, a histone H2B variant binds to a histone H2A variant called H2AZ, localizes to active genes, and supports transcription in those regions. In mice, a variant called H2BE helps control the expression of olfactory genes. This supports the idea that isoforms of histone H2B may have specialized functions in different tissues.
Isoforms.
There are sixteen variants of histone H2B found in humans, thirteen of which are expressed in regular body cells and three of which are only expressed in the testes. These variants, also called isoforms, are proteins that are structurally very similar to the regular histone H2B but feature some specific variations in their amino acid sequence. All variants of histone H2B contain the same number of amino acids, and the variations in sequence are few in number. Only two to five amino acids are changed, but even these small differences can alter the isoform’s, higher level structure.
Histone H2B isoforms interact differently with other proteins, are found in specific regions of chromatin, have different types and numbers of post-translational modifications, and are more or less stable than regular histone H2B. All of these differences accumulate and cause the isoforms to have unique functions and even function differently in different tissues.
Many histone H2B isoforms are expressed in a DNA replication independent manner. They are produced at the same level during all phases of the cell cycle. Regular histone H2B is only added to nucleosomes during the S-phase of the cell cycle when DNA is replicated; histone H2B isoforms can be added to nucleosomes at other times during the cell cycle. Histone variants of H2B can be explored using "HistoneDB with Variants" database.
Post-Translational Modifications.
Histone H2B is modified by a combination of several types of post-translational modifications. These modifications affect the structural and functional organization of chromatin, and the majority of them are found outside the globular domain of the nucleosome where amino acid residues are more accessible. Possible modifications include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. Acetylation, phosphorylation, and ubiquitination are the most common and most studied modifications of histone H2B.
Acetylation.
Histone H2B proteins found both in the promoter and coding regions of genes contain specific patterns of hyperacetylation and hypoacetylation on certain lysine residues found in the N-terminal tail. Acetylation relies on specific histone acetyltransferases that work at gene promoters during transcriptional activation. Adding an acetyl group to lysine residues in one of several positions in the amino acid tail contributes to the activation of transcription. In fact, scientists consider acetylation of histone H2B’s N-terminal tails, such as H2BK5ac, to be an extremely important part of regulating gene transcription.
"O"-GlcNAcylation.
Modification of H2B S112 with "O"-GlcNAc is thought to facilitate monoubiquitination of K112, which in turn is associated with transcriptionally activated regions.
Phosphorylation.
In histone H2B, a phosphorylated serine or threonine residue activates transcription. When a cell experiences metabolic stress, an AMP-activated protein kinase phosphorylates the lysine at position 36 in histone H2B of the promoter and coding regions on DNA, which helps regulate transcriptional elongation. If cells receive multiple apoptotic stimuli, caspase-3 activates the Mst1 kinase, which phosphorylates the serine at position 14 in all histone H2B proteins, which helps facilitate chromatin condensation. DNA damage can induce this same response on a more localized scale very quickly to help facilitate DNA repair.
Ubiquitination.
Ubiquitin residues are usually added to the lysine at position 120 on histone H2B. Ubiquitinating this lysine residue activates transcription. Scientists have discovered other ubiquitination sites in recent years, but they are not well studied or understood at this time. Ubiquitin-conjugating enzymes and ubiquitin ligases regulate the ubiquitination of histone H2B. These enzymes use co-transcription to conjugate ubiquitin to histone H2B. Histone H2B’s level of ubiquitination varies throughout the cell cycle. All ubiquitin moieties are removed from histone H2B during metaphase and re-conjugated during anaphase.
Genetics.
Histone H2B’s amino acid sequence is highly evolutionarily conserved. Even distantly related species have extremely similar histone H2B proteins. The histone H2B family contains 214 members from many different and diverse species. In humans, histone H2B is coded for by twenty-three different genes, none of which contain introns. All of these genes are located in histone cluster 1 on chromosome 6 and cluster 2 and cluster 3 on chromosome 1. In each gene cluster, histone H2B genes share a promoter region with sequences that code for histone H2A. While all genes in the histone cluster are transcribed at high levels during S-phase, individual histone H2B genes are also expressed at other times during the cell cycle. They are dually regulated by the cluster promoter sequences and their specific promoter sequences.


Obtusifoliol

Obtusifoliol is a metabolic intermediate of sterols made by certain fungis, can be converted to Delta8,14-Sterol by enzyme ERG11 (CYP51F1).


Catgrip

Catgrips are small cation-binding molecular features of proteins and peptides. Each consists of the main chain atoms only of three consecutive amino acid residues. The first and third main chain CO groups bind the cations, often calcium, magnesium, potassium or sodium, with no side chain involvement. Many catgrips bind a water molecule instead of a cation; it is hydrogen-bonded to the first and third main chain CO groups. Catgrips are found as calcium-binding features in annexins, matrix metalloproteinases (e.g.serralysins), subtilisins and phospholipase A2. They are also observed in synthetic peptides and in cyclic hexapeptides made from alternating D,L amino acids.
The conformation of a catgrip is such that the CO groups of the first and third amino acid residues are liable to be electrostatically attracted or hydrogen bonded to a positively charged, or partially positively charged, atom or group. Catgrips are defined by the phi, psi main chain dihedral angles of the second and third amino acids in the nest.
If two catgrips overlap such that two successive residues are both residue i of catgrips, a compound catgrip is formed. If three overlap such that three successive residues are residue i of catgrips, a longer catgrip is formed and so on. Such overlapping motifs may bind one, two or three metal ions. An example is given by residues 586-590 of Cas9 endonuclease (pdb code 4oge) where the mainchain CO groups from residues i, i+2 and i+4 bind a magnesium ion.
Another tripeptide motif that often binds cations or δ+ groups via main chain CO groups is called the niche (protein structural motif).


Mothers against decapentaplegic homolog 7

Mothers against decapentaplegic homolog 7 or SMAD7 is a protein that in humans is encoded by the "SMAD7" gene.
SMAD7 is a protein that, as its name describes, is a homolog of the Drosophila gene: "Mothers against decapentaplegic". It belongs to the SMAD family of proteins, which belong to the TGFβ superfamily of ligands. Like many other TGFβ family members, SMAD7 is involved in cell signalling. It is a TGFβ type 1 receptor antagonist. It blocks TGFβ1 and activin associating with the receptor, blocking access to SMAD2. It is an inhibitory SMAD (I-SMAD) and is enhanced by SMURF2.
Smad7 enhances muscle differentiation.
Structure.
Smad proteins contain two conserved domains. The Mad Homology domain 1 (MH1 domain) is at the N-terminal and the Mad Homology domain 2 (MH2 domain) is at the C-terminal. Between them there is a linker region which is full of regulatory sites. The MH1 domain has DNA binding activity while the MH2 domain has transcriptional activity. The linker region contains important regulatory peptide motifs including potential phosphorylation sites for mitogen-activated protein kinases(MAPKs), Erk-family MAP kinases, the Ca2+ /calmodulin-dependent protein kinase II (CamKII) and protein kinase C (PKC). Smad7 does not have the MH1 domain. A proline-tyrosine (PY) motif presents at its linker region enables its interaction with the WW domains of the E3 ubiquitin ligase, the Smad ubiquitination-related factors (Smurf2). It resides predominantly in the nucleus at basal state and translocates to the cytoplasm upon TGF-β stimulation.
Function.
SMAD7 inhibits TGF-β signaling by preventing formation of Smad2/Smad4 complexes which initiate the TGF-β signaling. It interacts with activated TGF-β type I receptor therefore block the association, phosphorylation and activation of Smad2. By occupying type I receptors for Activin and bone morphogenetic protein (BMP), it also plays a role in negative feedback of these pathways.
Upon TGF- β treatment, Smad7 binds to discrete regions of Pellino-1 via distinct regions of the Smad MH2 domains. The interaction blocks the formation of the IRAK1-mediated IL-1R/TLR signaling complex therefore abrogates NF-κB activity, which subsequently causes reduced expression of pro-inflammatory genes.
While Smad7 is induced by TGF-β, it is also induced by other stimuli, such as epidermal growth factor (EGF), interferon-γ and tumor necrosis factor (TNF)-α. Therefore, it provides a cross-talk between TGF-β signaling and other cellular signaling pathways.
Role in cancer.
A mutation located in SMAD7 gene is a cause of susceptibility to colorectal cancer (CRC) type 3. Perturbation of Smad7 and suppression of TGF-β signaling was found to be evolved in CRC. Case control studies and meta-analysis in Asian and European populations also provided evidence that this mutation is associated with colorectal cancer risk.
TGF-β is one of the important growth factors in pancreatic cancer. By controlling the TGF-β pathway, smad7 is believed to be related to this disease. Some previous study showed over-expression of Smad7 in pancreatic cells but there was a recent study showed a low Smad7 expression. The role of Smad7 in pancreatic cancer is still controversial.
Over-expression or constitutive activation of epidermal growth factor receptor (EGFR) can promote tumor processes. EGF-induced MMP-9 expression enhances tumor invasion and metastasis in some kinds of tumor cells such as breast cancer and ovarian cancer. Smad7 exerts an inhibitory effect on the EGF signaling pathway. Therefore, it may play a role in prevention of cancer metastasis.
Use in Pharmacology.
SMAD7 signaling has been studied in a recent Celgene Phase III trial, NCT ID number 94, which interacts with the SMAD7 pathway. This drug (Mongersen) was studied in patients with Crohn's disease.
Interactions.
Mothers against decapentaplegic homolog 7 has been shown to interact with:


Cholesterol 7alpha-monooxygenase

In enzymology, a cholesterol 7alpha-monooxygenase () is an enzyme that catalyzes the chemical reaction
The 4 substrates of this enzyme are cholesterol, NADPH, H+, and O2, whereas its 3 products are 7-alpha-hydroxycholesterol, 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 cholesterol,NADPH:oxygen oxidoreductase (7alpha-hydroxylating). Other names in common use include cholesterol 7alpha-hydroxylase, and CYP7A1. This enzyme participates in bile acid biosynthesis and ppar signaling pathway. It employs one cofactor, heme.


Gamma-glutamyltranspeptidase

Gamma-glutamyltranspeptidase may refer to:


Holin superfamily II

The Holin superfamily II is a superfamily of putative pore-forming proteins. It is one of the seven different holin superfamilies in total. In general, these proteins are thought to play a role in regulated cell death, although functionality varies between families and individual members. The Holin superfamily II includes the TC families:
All four of these families are derived from Proteobacteria and are of relatively small sizes, the average size of the proteins within the entire superfamily is 78 ± 14 amino acyl residues (aas). Some exceptions can be found (i.e., Lysis S family protein with 720 aas, TC# 1.E.1.1.7). As in holin superfamily I, proteins in these families generally exhibit of 2 transmembrane spanners (TMSs). Families 1 and 6 in Superfamily II are recognized domains in the Conserved Domains Database (CDD) as Lysis S and PHA00426 superfamilies, respectively, while families 7, and 25 lack recognized conserved domains.


Hexosamines

Hexosamines are amino sugars created by adding an amine group to a hexose.
Examples include:


Talazoparib

Talazoparib, sold under the brand name Talzenna, is an orally available poly ADP ribose polymerase (PARP) inhibitor developed by Pfizer for the treatment of advanced breast cancer with germline BRCA mutations. Talazoparib is similar to the first in class PARP inhibitor, olaparib. It was approved in October 2018, in the United States and June 2019, in the European Union for germline BRCA-mutated, HER2-negative locally advanced or metastatic breast cancer.
Side effects.
The most serious side effects in studies were related to the blood forming system and included anaemia (low red blood cell count), neutropenia (low neutrophil blood cell count) and thrombocytopenia (low platelet count). Serious forms of these conditions (grade 3 to 4) occurred in 39%, 21% and 15% of patients, respectively. Other adverse effects such as headache, nausea, hair loss and fatigue were mostly mild.
Interactions.
Combination with drugs that inhibit P-glycoprotein or BCRP may increase talazoparib concentrations in the body.
Mechanism of action.
Talazoparib acts as an inhibitor of poly ADP ribose polymerase (PARP) which aids in single strand DNA repair. Cells that have BRCA1/2 mutations are susceptible to the cytotoxic effects of PARP inhibitors because of an accumulation of DNA damage. Talazoparib is theorized to have a higher potency than olaparib due to the additional mechanism of action called PARP trapping. PARP trapping is the mechanism of action where the PARP molecule is trapped on the DNA, which interferes with the cells ability to replicate. Talazoparib is found to be ~100 fold more efficient in PARP trapping than olaparib. However, this increased potency may not translate directly to clinical effectiveness as many other factors must be considered.
Commercialization.
Talazoparib was originally developed by BioMarin Pharmaceutical Inc. However, Medivation Inc. acquired all worldwide rights to talazoparib in August 2015, to expand their global oncology franchise. Medivation acquired talazoparib for $410 million with additional payments of up to $160 million in royalties and milestones. Under this agreement, Medivation assumed all financial responsibilities for the continued development, regulatory, and commercialization of talazoparib.


BLUF domain

In molecular biology, the BLUF domain (sensors of blue-light using FAD) is a FAD-binding protein domain. They are present in various proteins, primarily from bacteria, for example a BLUF domain is found at the N-terminus of the AppA protein from "Rhodobacter sphaeroides". The BLUF domain is involved in sensing blue-light (and possibly redox) using FAD and is similar to the flavin-binding PAS domains and cryptochromes. The predicted secondary structure reveals that the BLUF domain has a novel FAD-binding fold.


Biliverdin reductase

Biliverdin reductase (BVR) is an enzyme () found in all tissues under normal conditions, but especially in reticulo-macrophages of the liver and spleen. BVR facilitates the conversion of biliverdin to bilirubin via the reduction of a double-bond between the second and third pyrrole ring into a single-bond.
There are two isozymes, in humans, each encoded by its own gene, biliverdin reductase A (BLVRA) and biliverdin reductase B (BLVRB).
Mechanism of catalysis.
BVR acts on biliverdin by reducing its double-bond between the pyrrole rings into a single-bond. It accomplishes this using NADPH + H+ as an electron donor, forming bilirubin and NADP+ as products.
BVR catalyzes this reaction through an overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 as key residues. This binding site attaches to biliverdin, and causes its dissociation from heme oxygenase (HO) (which catalyzes reaction of ferric heme --> biliverdin), causing the subsequent reduction to bilirubin.
Structure.
BVR is composed of two closely packed domains, between 247-415 amino acids long and containing a Rossmann fold. BVR has also been determined to be a zinc-binding protein with each enzyme protein having one strong-binding zinc atom.
The C-terminal half of BVR contains the catalytic domain, which adopts a structure containing a six-stranded beta-sheet that is flanked on one face by several alpha-helices. This domain contains the catalytic active site, which reduces the gamma-methene bridge of the open tetrapyrrole, biliverdin IX alpha, to bilirubin with the concomitant oxidation of a NADH or NADPH cofactor.
Function.
BVR works with the biliverdin/bilirubin redox cycle. It converts biliverdin to bilirubin (a strong antioxidant), which is then converted back into biliverdin through the actions of reactive oxygen species (ROS). This cycle allows for the neutralization of ROS, and the reuse of biliverdin products. Biliverdin also is replenished in the cycle with its formation from heme units through heme oxygenase (HO) localized from the endoplasmic reticulum.
Bilirubin, being one of the last products of heme degradation in the liver, is further processed and excreted in bile after conjugation with glucuronic acid. In this way, BVR is essential in many mammals for the disposal of heme catabolites – especially in the fetus where the placental membranes are bilirubin-permeable but not biliverdin-permeable - aiding in the removal of potentially toxic protein build-up.
BVR has also more recently been recognized as a regulator of glucose metabolism and in cell growth and apoptosis control, due to its dual-specificity kinase character. This control over glucose metabolism indicates that BVR may play a role in pathogenesis of multiple metabolic diseases - the notable one being diabetes, by control of the upstream activator of insulin growth factor-1 (IGF-1) and mitogen-activated protein kinase (MAPK) signaling pathway.
Disease relevance.
BVR acts as a means to regenerate bilirubin in a repeating redox cycle without significantly modifying the concentration of available bilirubin. With these levels maintained, it appears that BVR represents a new strategy for the treatment of multiple sclerosis and other types of oxidative stress-mediated diseases. The mechanism is due to the amplification of the potent antioxidant actions of bilirubin, as this can ameliorate free radical-mediated diseases.
Studies have shown that the BVR redox cycle is essential in providing physiological cytoprotection. Genetic knock-outs and reduced BVR levels have demonstrated increased formation of ROS, and results in augmented cell death. Cells that experienced a 90% reduction in BVR experienced three times normal ROS levels. Through this protective and amplifying cycle, BVR allows low concentrations of bilirubin to overcome 10,000-fold higher concentrations of ROS.


Negative elongation factor

In molecular biology, the NELF (negative elongation factor) is a four-subunit protein complex (NELF-A, NELF-B, NELF-C/NELF-D, and NELF-E) that negatively impacts transcription by RNA polymerase II (Pol II) by pausing about 20-60 nucleotides downstream from the transcription start site (TSS).
Structure.
The NELF has four subunits within its complex which are the following: NELF-A, NELF-B, NELF-C/NELF-D, and NELF-E. The NELF-A subunit is encoded by the gene WHSC2 (Wolf-Hirschhorn syndrome candidate 2). Micro-sequencing analysis demonstrated that NELF-B was the protein previously identified as being encoded by the gene COBRA1. It is unknown whether or not NELF-C and NELF-D are peptides resulting from the same mRNA with different translation initiation sites; possibly differing only in an extra 9 amino acids for NELF-C at the N-terminus, or peptides from different mRNAs entirely. A single NELF complex consists of either NELF-C or NELF-D, but not both. NELF-E is also known as RDBP.
Function and Interactions.
NELF is located in the nucleus. NELF binds in a stable complex with DSIF (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-sensitivity inducing factor) and RNA polymerase II together, but not with either alone. Due to its role in transcription, NELF is also a key player in the negative function of DSIF.NELF also works with DSIF to inhibit the speed of Pol II during the elongation phase in transcription. In "D. melanogaster", the HSP70 gene is affected by NELF and DSIF through the induction of promoter proximal pausing. It is thought that NELF arose to assist DSIF by amplifying its negative effects in order to increase gene expression control. P-TEFb (positive transcription elongation factor b) inhibits the effect of NELF and DSIF on Pol II elongation, via its phosphorylation of serine-2 of the C-terminal domain of Pol II, and the SPT5 subunit of DSIF, causing dissociation of NELF. However, many mechanisms by which NELF and DSIF operate remain unclear. NELF homologues exist in some metazoans (e.g. insects and vertebrates) but have not been found in plants, yeast, or nematodes (worms).
Interactions by subunit:
NELF-A: Pol II complex.
NELF-B: KIAA1191, NELF-E, and an early sequence of BRCA1.
NELF-C/D: ARAF1, PCF11, and KAT8.
NELF-E: NELF-B and HIV TAR RNA.
NELF undergoes Phase separation in vitro and Condensation in vivo
Clinical Significance.
The NELF complex is also possibly a player in the enlistment of gene PCF11 to the stopped Pol II in HIV-1 latency. NELF-A may play a role in the phenotype of Wolf-Hirschhorn syndrome (WHS) as it is mapped to the critical area of deletion on the short arm of chromosome 4. Pol II pausing controlled by NELF is a key source of R-loop aggregation in mammary epithelial cells that are BRCA1-deficient, which could ultimately lead to tumorigenesis.


Guaijaverin

Guaijaverin is the 3-O-arabinoside of quercetin. It is found in the leaves of "Psidium guajava", the common guava.


Multi-antimicrobial extrusion protein

Multi-antimicrobial extrusion protein (MATE) also known as multidrug and toxin extrusion or multidrug and toxic compound extrusion is a family of proteins which function as drug/sodium or proton antiporters.
Function.
The MATE proteins in bacteria, archaea and eukaryotes function as fundamental transporters of metabolic and xenobiotic organic cations.
Structure.
These proteins are predicted to have 12 alpha-helical transmembrane regions, some of the animal proteins may have an additional C-terminal helix. The X-ray structure of the NorM was determined to 3.65 Å, revealing an outward-facing conformation with two portals open to the outer leaflet of the membrane and a unique topology of the predicted 12 transmembrane helices distinct from any other known multidrug resistance transporter.
Discovery.
The multidrug efflux transporter NorM from "V. parahaemolyticus" which mediates resistance to multiple antimicrobial agents (norfloxacin, kanamycin, ethidium bromide etc.) and its homologue from "E. coli" were identified in 1998. NorM seems to function as drug/sodium antiporter which is the first example of Na+-coupled multidrug efflux transporter discovered. NorM is a prototype of a new transporter family and Brown "et al". named it the multidrug and toxic compound extrusion family. NorM is nicknamed "Last of the multidrug transporters" because it is the last multidrug transporter discovered functionally as well as structurally.
Genes.
The following human genes encode MATE proteins:


Actin remodeling

Actin remodeling is the biochemical process that allows for the dynamic alterations of cellular organization. The remodeling of actin filaments occurs in a cyclic pattern on cell surfaces and exists as a fundamental aspect to cellular life. During the remodeling process, actin monomers polymerize in response to signaling cascades that stem from environmental cues. The cell's signaling pathways cause actin to affect intracellular organization of the cytoskeleton and often consequently, the cell membrane. Again triggered by environmental conditions, actin filaments break back down into monomers and the cycle is completed.
Actin-binding proteins (ABPs) aid in the transformation of actin filaments throughout the actin remodeling process. These proteins account for the diverse structure and changes in shape of Eukaryotic cells. Despite its complexity, actin remodeling may result in complete cytoskeletal reorganization in under a minute.
Structural composition of actin.
Actin remains one of the most abundant proteins in all of Eukarya and is an enzyme (ATPase) that gradually hydrolyzes ATP. It exists in two forms within eukaryotic cells: globular or G-actin and filament/filamentous or F-actin. Globular actin is the monomeric form of the protein while the filamentous actin is a linear polymer of globular subunits. The assembly of filamentous actin arises as a result of weak, noncovalent interactions between G-actin and appears in the arrangement of a two-stranded asymmetrical helical polymer.
The asymmetrical nature of F-actin allows for distinct binding specificities at each terminus. The terminus that presents an actin subunit with an exposed ATP binding site is commonly labeled the "(-) end". Whereas, the opposite end of the polymer that presents a cleft and lacks a free ATP binding site is referred to as the "(+) end". Additionally, the respective ends of the actin microfilament are often specified by their appearance under transmission electron microscopy during a technique known as "decoration", where the addition of myosin results in distinctive actin-myosin binding at each terminus. The terms "pointed end" and "barbed end" refer to the "(-) end" and "(+) end" respectively.
Within the cell, the concentrations of G-actin and F-actin continuously fluctuate. The assembly and disassembly of F-actin is regularly known as "actin tread-milling". In this process, G-actin subunits primarily add to the "barbed end" of the filamentous polymer. This end proves to be both more thermodynamically favored for the addition of G-actin and kinetically dynamic as well. Simultaneously, older G-actin monomers "fall off" of the pointed end of the microfilament. At the "pointed end" of the F-actin polymer, actin monomers are bound to ADP, which dissociates more readily and rapidly than ATP-bound actin, which is found at the "barbed end" of the polymer. Thus, in environments with high concentrations of free actin subunits, filamentous growth at the "barbed end" remains greater than that of the "pointed end". This "tread-milling", essentially exists as a simplified explanation of the actin remodeling process.
Actin remodeling cycle.
Cell surface (cortical) actin remodeling is a cyclic (9-step) process where each step is directly responsive to a cell signaling mechanism. Over the course of the cycle, actin begins as a monomer, elongates into a polymer with the help of attached actin-binding-proteins, and disassembles back into a monomer so the remodeling cycle may commence again. The dynamic function of actin remodeling is directly correlated to the immense variability of cell shape, structure, and behavior.
Initiation
Consists of a number of different possible mechanisms that ultimately determine where and when actin filament elongation is to occur. In the mechanism that involves the uncapping of the barbed-end, diffusion-regulated actin polymerization of subunits bound to actin-monomer-sequestering proteins control initiation. Thymosin and Profilin both exist as actin-monomer-sequestering proteins that maintain the ability to limit spontaneous nucleation from occurring, thus halting the actin remodeling process and returning the cycle to its first step. Additionally, the cell utilizes polyphosphoinositides to aid in the removal of all known "barbed end" capping proteins.
"Possible Mechanisms:"
Elongation
Facilitated "in vivo" by polymerization promoters and barbed-end capping inhibitory proteins. The elongation phase begins when the concentration of short, F-actin polymers is significantly larger than at equilibrium. At this point, both termini accept the addition of new monomers (although primarily at the "barbed end") and the actin microfilament lengthens.
Termination
Involves the degradation of polyphosphoinositides and reactivation of "barbed end" capping proteins Hsp70 and CapZ, thereby reinitiating barbed-end capping and greatly diminishing elongation. Despite the presence of active capping proteins, certain inhibitors including profilin, formins, ENA and VASP promote elongation. These inhibitors may function in a variety of different methods, however, most employ the inhibition of subunit depolymerization and actin-depolymerizing actin-binding-proteins.
Branching amplification
Consists of the nucleation of new actin microfilaments from the existing sides of F-actin. The cell employs Arp2/3 complex to temporarily bind to existing polymers at a 70° angle. The Arp2/3 complex then elongates into a filamentous branch that proves essential for intracellular reorganization through cytoskeletal changes. This change in infrastructure may alter cell shape and behavior and is often used to transport vesicles, pathogens, or other related structures.
Actin filament crosslinking
Results in the overall stabilization of the actin filament network. The cell utilizes crosslinking proteins are various sizes to accomplish different means of stability within the binding network. Relatively small ABP's such as scruin, fimbrin, and espin function by solidifying actin filament bundles. Larger ABPs that exhibit coil-like qualities such as filament function in the promotion of orthogonal organization. As a whole, actin crosslinking provides framework for which the cell may transport signaling intermediates needed for other steps within the actin remodeling cycle.
Actin filament contraction and cargo motoring
Represents the ability for the actin filament network to react to environmental conditions and respond through various forms of vesicle and signal trafficking. Most commonly, the myosin protein exists as a "motor" that escorts cellular "cargo" throughout the cell. Myosin, primarily Myosin II, is also essential to the generation of contractile forces amongst the actin filaments.
Membrane attachment to actin network
Attachment of the actin-orthogonal network to the cell's membrane proves essential to the locomotion, shape, and mechanical function of the cell. The dynamic nature of a cell remains directly related to the actin-filament network's ability to respond to the contractile forces that result from environmental and internal cues.
Actin filament disassembly
The immobilization by interpenetration of actin filaments results from two distinct ABP families. The gelsolin protein family is believed to be the most efficient in the disruption of actin filaments and is considered a "strong severing protein". These proteins respond to an increase in Ca2+ and cap the "barbed end" of the recently severed F-actin. The increased level of Ca2+ may also destabilize the actin-filament network by interfering with the binding of crosslinking proteins. The ADF/Cofilin protein family also serves to severe actin-filament networks through the weak severing of actin networks. This form of weak severing does not tightly cap the "barbed ends" but does allow for the disassociation of actin monomers and thus the disassembly of F-actin.
Monomer sequestration that prevents spontaneous nucleation
Exists as the turnover point in the actin remodeling cycle. The proteins thymosin and profilin prevent the spontaneous nucleation of new actin trimers. The absence or inhibition of these proteins results in the cell's ability to commence the actin remodeling cycle and produce elongated F-actin.