ALK inhibitor

ALK inhibitors are anti-cancer drugs that act on tumours with variations of anaplastic lymphoma kinase (ALK) such as an EML4-ALK translocation. They fall under the category of tyrosine kinase inhibitors, which work by inhibiting proteins involved in the abnormal growth of tumour cells. All the current approved ALK inhibitors function by binding to the ATP pocket of the abnormal ALK protein, blocking its access to energy and deactivating it.
A majority of ALK-rearranged NSCLC harbour the EML4-ALK fusion, although as of 2020, over 92 fusion partners have been discovered in ALK+ NSCLC. For each fusion partner, there can be several fusion variants depending on the position the two genes were fused at, and this may have implications on the response of the tumour and prognosis of the patient.
Approved inhibitors.
First generation.
Crizotinib (also a ROS1 and c-MET inhibitor) was approved in Aug 2011 by the US FDA for ALK-positive NSCLC. At the time of the discovery of ALK translocations as a molecular driver in NSCLC, crizotinib was being investigated by Pfizer as a potential c-MET inhibitor. Its activity against ALK being known, Pfizer shifted its investigations to focus on this indication, and obtained a full approval 4 years later. Crizotinib's efficacy was proven in phase III trial, PROFILE 1007, when it was compared to then-standard second-line pemetrexed or docetaxel chemotherapy. It induced tumour stabilisation or shrinkage in 90% of patients. Its lack of penetrance in the brain and non-optimal specificity for ALK meant resistance mostly arose within a year, with the brain being a common site of progression.
Second generation.
Despite the excitement of crizotinib's therapeutic success, there was a need to conceive new drugs with better brain penetrance, higher specificity and targeting a broader set of resistance mutations.
As such, Novartis' ceritinib was approved by the FDA in April 2014 for treatment of NSCLC. It provided good brain penetrance and a significant progression-free survival benefit against chemotherapy in the first line as demonstrated in the ASCEND-4 trial.
Roche's alectinib was FDA approved Dec 2015 (accelerated) for patients who had progressed on crizotinib, with full approval in 2017 as a first-line treatment for ALK-positive NSCLC. Like ceritinib, it provided excellent brain penetrance and high response rates. It demonstrated a clear benefit against both first-line chemotherapy and first-line crizotinib. This was based on the phase 3 ALEX trial comparing it with crizotinib.
Ariad's and Takeda's brigatinib (also an inhibitor of mutated EGFR) was the latest second-generation inhibitor and was approved in April 2017 by the US FDA for ALK-positive NSCLC. It is very similar to alectinib in efficacy, while being active against some resistant mutations such as the common G1202R mutation that provides resistance to alectinib.
Third generation.
Pfizer's lorlatinib was the first third-generation inhibitor and was approved in 2018 by the US FDA for ALK-positive NSCLC after progression on a first or second-generation inhibitor. Its macrocyclic structure was designed specifically to address some of the most recalcitrant resistance mutations. Still, most tumours eventually develop resistance through various mechanisms, namely compound-mutations (two or three mutations simultaneously) or activation of alternative pathways, such as the c-MET pathway.
Clinical trials.
Additional ALK inhibitors currently (or soon to be) undergoing clinical trials include:
Discontinued
Investigational combinations.
While the response to ALK inhibitors is often very encouraging in patients with ALK+ NSCLC and lasts for a relatively long time, most of them eventually develop resistance, either through mutations in the ATP binding pocket or activation of alternative oncogenic pathways. Much research is being carried out on understanding the ways the cancer adapts and on how to reverse or delay resistance.
MEK pathway.
The MEK pathway (short for MAPK/ERK-Kinase) has been extensively shown to be critical for the survival of tumour cells subjected to ALK inhibition. Inhibition of this pathway was shown to enhance response and delay the onset of resistance in preclinical models. As of 2020, three clinical trials are running to test the following combinations of ALK inhibitors with MEK inhibitors: brigatinib+binimetinib, ceritinib+trametinib, and alectinib+cobimetinib. Results for the last two are expected around 2020-2021.
EGFR/HER2 pathway.
The EGFR and HER2 pathways are commonly abnormally activated in a large proportion of cancers. This was shown to be the case in preclinical models of ALK+ NSCLC subjected to ALK inhibition, both "in vitro" and "in vivo". Surprisingly, cells were only sensitive to EGFR/HER2 inhibition when in the process of adapting to ALK inhibitors: both naïve cells and fully-adapted cells showed no measurable response to EGFR/HER2 inhibition alone. Currently, solid preclinical studies have been carried out with second-generation inhibitors combined with afatinib, erlotinib and lapatinib. In all cases, the responses were enhanced by the combination with respect to monotherapy, but seemed more pronounced in afatinib and lapatinib (dual EGFR/HER2 inhibitors) than in erlotinib (which only inhibits EGFR).
Anti-VEGF therapies.
Several trials are investigating the combination of anti-VEGF antibody bevacizumab with ALK inhibitors such as alectinib and brigatinib. Bevacizumab is an antiangiogenic antibody, which normalizes the complex blood vessel structures around cancer and prevents new blood vessels from forming, thus starving the tumour and preventing its proliferation.
Local consolidation therapy.
The use of either radiotherapy or surgery in addition to an ALK inhibitor is known as local consolidation therapy, and as of 2020, it is being investigated by three clinical trials. Their goal is to determine whether it delays resistance to the drugs compared with monotherapy. Some make use of SBRT (stereotactic body radiation therapy), a very precise radiation technique able to provide high doses with minimal side effects. Most trials of LCT in NSCLC focus on oligometastatic disease (under 3-5 lesions, depending on the definitions), but preliminary results of the BRIGHTSTAR trial indicate this method may be safe and well tolerated irrespective of the number of lesions.
NPM-ALK.
NPM-ALK is a different variation/fusion of ALK that drives anaplastic large-cell lymphomas (ALCLs) and is the target of other ALK inhibitors such as TAE-684.


3-hydroxyoctanoyl-(acyl-carrier-protein) dehydratase

In enzymology, a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase () is an enzyme that catalyzes the chemical reaction
Hence, this enzyme has one substrate, (3R)-3-hydroxyoctanoyl-(acyl-carrier-protein), and two products, oct-2-enoyl-[acyl-carrier-protein] and H2O.
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds. The systematic name of this enzyme class is (3R)-3-hydroxyoctanoyl-[acyl-carrier-protein] hydro-lyase (oct-2-enoyl-[acyl-carrier protein]-forming). Other names in common use include D-3-hydroxyoctanoyl-[acyl carrier protein] dehydratase, D-3-hydroxyoctanoyl-acyl carrier protein dehydratase, beta-hydroxyoctanoyl-acyl carrier protein dehydrase, beta-hydroxyoctanoyl thioester dehydratase, beta-hydroxyoctanoyl-ACP-dehydrase, and (3R)-3-hydroxyoctanoyl-[acyl-carrier-protein] hydro-lyase.


Hemagglutinin esterase

Hemagglutinin esterase (HEs) is a glycoprotein that certain enveloped viruses possess and use as invading mechanism. HEs helps in the attachment and destruction of certain sialic acid receptors that are found on the host cell surface. Viruses that possess HEs include "influenza C virus", toroviruses, and coronaviruses (but not SARS-like coronaviruses). HEs is a dimer transmembrane protein consisting of two monomers, each monomer is made of three domains. The three domains are: membrane fusion, esterase, and receptor binding domains.
The different HEs enzyme activities include: receptor binding activity, receptor hydrolysis (esterase) activity, and membrane fusion activity. The receptor binding activity involve the attachment of HEs to N-acetyl-9-O-acetylneuraminic acid (9-O-Ac- Neu5Ac) of glycolipids and glycoproteins and in turn serve as viral receptor. Receptor hydrolysis (esterase) activity allows virus particles to escape the infected cell by removing an acetyl group from the C9 position of terminal 9-O-Ac-Neu5Ac residues. Membrane fusion activity helps in incorporation viral genome into the host cell cytoplasm by enhancing the attachment between the viral envelope and host cell membrane.
In certain influenza viruses, the cell surface consists of both hemagglutinin (HA) and neuraminidase (NA) proteins that encompass enzymatic activities, whereas hemagglutinin-esterase fusion (HEF) proteins have been found to be the primary single spike protein that combines all of the enzymatic activities listed above. HEF proteins have been tested to be high-temperature and low-pH resistant and are the primary source of virulence in viruses. Influenza C have been shown to have unique HEF structure proteins that enhance its ability to infect the host cell compared to influenza A and B.
The folding of different domains in the hemagglutinin-esterase protein is important for intracellular transport of proteins from the endoplasmic reticulum to the Golgi apparatus. The presence of oligosaccharide chains in the E, F, and R domains of the HE enzyme also influence intracellular transport. Acylation of the hemagglutinin-esterase has shown to play an essential role in virus particle assembly replication. The exact process of enzyme catalytic cleavage has not yet been detailed out. However, proteolytic cleavage must occur before hemagglutinin-esterase membrane fusion activity. HEF proteins have a unique spikes hexagonal arrangement. This feature is unique to "influenza C virus" particles. The arrangement is a covering outside of the particle.
Structure.
Certain studies revealed that coronavirus and toroviruses HE was originated from HEF glycoprotein that is found in influenza C viruses which resulted from alteration of hemagglutinin esterase from a trimer into a dimer glycoprotein. During this process, the receptor destroying enzyme acetyl esterase domain stayed unchanged. However, the HE receptor binding domain has been altered in which that the ligand is bound in opposite orientation than before. Both coronavirus and toroviruses HE monomers are made up of the same three domains: central esterase/hydrolase domain, receptor binding lectin domain, and membrane proximal domain which is small. The two monomers of HE dimer in both CoV and ToV involve the same two contact regions (CR 1 and 2). CR 1 contain the receptor binding domain and contact region 2 that contain membrane proximal domain. Yet, ToV HE contacts region 2 contain additional esterase domain. As a result, the CR 2 surface is larger in ToV HEs than in CoV HEs.  However, close to the carboxylic terminal membrane anchor, there are number of disulfide bridges between Cys385 of coronavirus HE that in turn keep the HE dimers connected to each other.
In CoV HE, the two R domain beta sheets are connected to each other forming a continuous intermolecular beta sheet across the dimer interface. On the other hand, in ToV they are oriented at angles. As a result, the beta sheet of receptor binding domain in ToV is more twisted, the contact region 1 is smaller, and the R domains position are shifted along the Beta strands compared to CoV.
Crystalline structure.
"Initial studies using electron microscopy showed that the HEF spike forms a mushroom-shaped trimer consisting of a membrane-near stalk and a globular head".
Later studies were able to examine and show a higher resolution structure (4.5 Å) of the hemagglutinin esterase fusion trimer using X-ray crystallography of the bromelain-cleaved ectodomain. Both hemagglutinin and hemagglutinin esterase fusion protein are similar in terms of structure and the folding of individual segments. yet, only 12% amino acid are identical between HA and HEF. One significant difference between HE and HEF is the presence of an additional bulge in HEF globular domain (bottom part of the domain) which contains the esterase region. The receptor-binding region in both HA and HEF is found in the upper part of the domain and contain only HEF1 residues. The stalk is made of three 60 Å long α- helices that contain: all sequences of HEF2 sequence, and certain HEF1 residues which are N-terminal residues (1–40), and C-terminal residues (367–432).
The crystalline structure shows that the way that HEF binds to 9-O-Ac- Neu5Ac is the same as the way HA binds to Neu5Ac. The binding parts include an α-helix, a loop and an extended strand. There are hydrogen bonds between the amino acids (Tyr127, Thr170, Gly172, Tyr227 and Arg292) and the hydroxyl-groups of the ligand, and other residues form the structural support of the receptor binding site. A unique hydrophobic pocket is present in the HEF binding site that in turn accommodates the acetyl methyl group.
Activity.
Receptor binding activity.
Glycolipids and glycoproteins contain N-acetyl-9-O-acetylneuraminic acid (9-O-Ac- Neu5Ac) that serve as viral receptor in which HEF binds to. HEF can bind to its receptor whether or not 9-O-Ac-Neu5Ac is attached by an α-2,3 or α-2,6 linkage to the next galactosyl residue. However, host specificity can be affected by terminal N-acetylneuraminic acid (Neu5Ac) and the glycosidic linkage of Neu5Ac. "Influenza C virus" can recognize 9-O-Ac-Neu5Ac on the surface of different cells due to its unique receptor specificity.
Receptor hydrolysis (esterase) activity.
The receptor hydrolase activity of HEF aids in the release of virus particles from an infected cell using esterase enzyme that cleaves acetyl from the C9 position of terminal 9-O-Ac-Neu5Ac. The esterase activity of HEF which is part of serine hydrolase class includes a nucleophilic attack of the hydroxyl group (OH) of a serine amino acid, with the help of two other amino acids (histidine and aspartic acid), on the carbonyl group of the substrate. Basic histidine enhances the reactivity of serine by polarizing and deprotonating its hydroxyl group. Along with that, aspartic acid polarizes histidine.
X-ray crystallography of the crystalline structure of HEF showed that serine 57, aspartic acid 352 and histidine 355 are the important amino acids for the esterase activity. Also, early studies showed that mutation in Ser57 and His355 residues can completely stop the esterase activity of HEF.
Membrane fusion activity.
The membrane fusion activity between, the viral envelope and endocytic vesicles of host cell, is important to help the virus inject their genome into the cytoplasm of the cell. In order to activate membrane fusion, Cleaving the precursor proteins HEF0 and HA0 into the subunits into the subunits HEF1 and HEF2, then exposing these proteins to acidic pH must be done prior.
Acidic pH causes protonation of specific amino acids that initiate certain rearrangement of the proteins.  The protonated amino acid is found to be histidine while its pKa matches the pH of endosome. Studies showed that there is about 0.7 difference in the pH value that trigger the membrane fusion activity from strain to strain of both influenza A and C.
Conformational change in HEF structure that occur at low pH results in the separation of fusion peptide from its location at the lower part of the stalk and exposing the outer surface of the molecule, so it can be inserted into the endosomal membrane. Another conformational change occur which cause the bending of the ectodomain to push the fusion peptide toward the transmembrane region. As a result of that, the virus and endosomal membranes get closer, exchanging lipids with hemifusion. Then, opening of a fusion pore and eventually complete merger of both lipid bilayers.
Folding and intracellular transport.
The folding of the hemagglutinin esterase protein and the way that the domains of the protein assembles contribute the transport of membrane and secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Researchers found that trimerization occurs at a point before exiting the ER. The monomers of the HE protein are folded before assembling is possible. Before the hemagglutinin esterase can report to the Golgi, it must be extensively folded and assembled.
The structure of hemagglutinin-esterase contributes to the intracellular transport. The hemagglutinin-esterase (HE) glycoprotein of "influenza C virus" is composed of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain (R). The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Oligosaccharide chains in the domains influence intracellular transport. A study showed that it was evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144), is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant HEs lacking one of these three sites failed to undergo the trimer assembly. Oligosaccharides are needed to maintain esterase activity in the F and R domains. If any of the domains lack an oligosaccharide chain, cell surface expression will be affected. It was found that HE monomer have acetylesterase activity because they possessed full-enzyme activity despite lack of an oligosaccharide chain. Oligosaccharide chains are important for intracellular transport, but not for fusion activity. Thus, oligosaccharide chains do not really promote membrane fusion.
S-acylation and RAFT-localization.
Acylation of the hemagglutinin-esterase enzyme is necessary for virus replication of "influenza C virus". It was found that recombinant virus lacking the acylation site of HEF could be rescued, but viral titers were reduced by one log relative to wild type Flu C. The resulting virus particles have a regular protein composition and no changes in their morphology were obvious by electron microscopy, but their hemolytic activity is reduced indicating a defect in membrane fusion. This is in comparison to several HA protein subtypes that showed similar results.
The hemagglutinin-esterase-fusion protein has co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. The HEF protein of "influenza C virus" has only one stearate attached to a transmembrane cysteine. Whereas HA of influenza A and B virus are associated with membrane rafts, cholesterol- and sphingolipid-enriched nanodomains of the plasma membrane, HEF is thought to localize to the bulk phase of the plasma membrane.
Proteolytic cleavage.
The binding and cleavage properties of the "influenza C" virions hemagglutinin-esterase (CHE) protein for 9-"O"-acetyl groups on sialic acids have been used in various assays using whole virions.
Proteolytic cleavage must occur before any membrane fusion activity of HE because it enables the protein to become activated by low pH. HEF proteins from all "influenza C virus" strains contain a monobasic cleavage site and are in this respect similar to HAs from human, porcine, equine and low pathogenic avian influenza A viruses. Polybasic cleavage sites that are present in HA of highly pathogenic avian influenza A viruses and processed by the ubiquitous protease furin are not found in any HEF protein. Consequently, replication of "influenza C virus" is limited to the site of virus infection, the respiratory tract. Unlike other influenza viruses, "influenza C virus" does not spread to other tissues. Multiple replication cycles of "influenza C virus" in tissue culture are enabled by addition of trypsin, whereas embryonated eggs produce infectious virus with cleaved HEF.
The enzyme catalyzing proteolytic cleavage of HEF has not been identified so far, but since both HA and HEF can be cleaved by trypsin at similar concentrations "in vitro"(5~20 µg/mL) it seems likely that they are also activated by the same enzymes inside cells. It is very common that HA is compared to HEF in many contexts.
Regular arrangement of HE spikes in virus particles.
The only spike of "influenza C virus", the hemagglutinin‐esterase‐fusion glycoprotein (HEF) combines receptor binding, receptor hydrolysis and membrane fusion activities. Like other hemagglutinating glycoproteins of influenza viruses HEF is S‐acylated, but only with stearic acid at a single cysteine located at the cytosol‐facing end of the transmembrane region. This HE protein however, has spikes in its structural organization as well.
HEF trimers on the surfaces of both spherical and filamentous particles are arranged in a reticular structure that has been described to consist mainly of hexagons. This feature is unique to "influenza C virus" particles. Even when HEF is removed from the membrane, the polymeric reticular structure that it originally had can still be seen. These results indicate that the hexagonal arrangement is an intrinsic feature of HEF and does not require other viral proteins such as M1 and that its formation likely involves lateral interaction between the ectodomains of HEF. The formation of the spike arrangement in virus particles acts like a coat around the virus particle by creating and covering it. This is similar to the hydrophobic effect in lipid bilayer membranes where nonpolar molecules and in the interior.
Location of N-glycosylation sites.
HEF N-glycosylation sites are located in figure 1. One sequon is located in HEF2 and six in HEF1. There are three in the globular head and 2 in the hinge region that connects the stalk with the head. The site at position 589 is not glycosylated because it is too close to the membrane-spanning region and cannot be accessed by the oligosaccharide transferase. Glycosylation is crucial for proper folding because it protects it from proteolytic degradation from the host cell and is important for the presentation of antigenic epitopes.
In influenza C.
The primary structure of HEF in influenza C contains 641 amino acids. It is a typical type 1 transmembrane protein with a short N-terminal, cleavable signal peptide, a long ectodomain, a transmembrane region and a very short cytoplasmic tail. HEF is composed of two subunits, HEF1 consisting of the N-terminal and HEF2 consisting of the transmembrane domain and the cytoplasmic tail. Electron microscopy analyzing the crystal structure of HEF showed that the spike of HEF forms a mushroom-shaped trimer consisting of a membrane-near stalk and a globular head. HEF contains only asparagine-linked carbohydrates which indicates that O-glycosylation does not occur. The location of the individual glycosylation sites in the crystal structure are located on seven of the eight highly conserved N-glycosylation sequons; one is located in the subunit, HEF2 and the other six are located on the subunit HEF1. Three sites are in the globular head and two are in the hinge region that connects the stalk with the head. There is a site at position 589 on the crystallized structure that is not glycosylated and this may be due close location to the membrane-spanning regions and cannot be accessed by oligosaccharide transferase. The positions of HA in influenza A are quite similar to influenza C by the majority of its carbohydrate positions being located in the larger subunit.
Location of intramolecular disulfide bonds.
In HEF1, 12/15 cysteine residues form 6 intrachain disulfide linkages that stabilizes the globular head domain. There are two cysteine residues, Cys373 and Cys399 that do not form disulfide linkages in the mature protein. They are located at the hinge that connects the globular head with the stalk region. The rest of the cysteine residues form interchain disulfide bonds with HEF in the ectodomain area, near the bottom of the trimer. These disulfide bonds in HEF2 allows the subunit to perform large conformational changes that catalyze membrane fusion.
In influenza viruses.
In influenza C, there are 15 cysteine residues in subunit HEF1, 12 of the residues form six intrachain disulfide links that stabilize the globular head domain. Two of the cysteine residues are not required for proper folding and function of HEF and/or they do not form a disulfide linkage in the mature protein located at the connection hinge. The remaining cysteine rescues forms an interchain disulfide bond with the only cysteine residue in the ectodomain of subunit HEF2. This residue is located at the bottom of the trimer. In comparison, influenza A, has similar disulfide bond distributions with one bond connecting HA1 with HA2, the majority are intrachain bonds. The rare occurrence of disulfide bonds in HEF2 and HA2 subunits allows these subunits to perform large conformational changes that catalyze membrane fusion.
Co- and post-translation modification.
During translocation of HEF into the lumen of the ER, the N-terminal signal peptide is cleaved, and carbohydrates are attached. Disulfide bond linkages are formed and remodeled. These modifications affect the folding and trimerization of the molecule. These processes are prerequisites for exiting cargo form the ER. Later on, a fatty acid chain is attached to the cysteine located on the end of the transmembrane region and HEF is cleaved into 2 subunits, this process is essential for virus replication.
In influenza viruses.
In comparison, influenza A, B, and C have different spike proteins, the haemagglutinin and the neuraminidase. The HEF surface glycoprotein of influenza C consists of three activities, receptor-binding, receptor-inactivating, and fusion activity. Receptor-binding mediates the attachment of the virus to N-acetyl-9-O-acetylneuraminic acid on the cell surface, receptor-inactivating releases the 9-O-acetyl group from N-acetyl-9-O-acetylneuraminic acid and the fusion activity depends on the post-translational proteolytic cleavage of HEF into two subunits as well as exposure to an acidic environment. In low pH conditions, a conformational change of HEF occurs. In influenza A, the rearrangement of hydrophobic sequences at the N-terminus of subunit HEF2 becomes exposed and induces the fusion of the viral envelope with the membrane of the target cell. Another way that fuses the viral envelope to the host cell is with endocytic vesicles. HEF does not cleave the terminal silica acid residue from carbohydrates but removes the acetyl group from the position C9 of N-acetyl-9-O-acetylneuraminic acid. This is required to release fresh budded virus particles from infected cells, which otherwise would be trapped in the plasma membrane if the receptor is still present
Influenza C is distinguishable from influenza A and B by its structural components. There are three amino acids that comprises the cytoplasmic portion of HEF, arginine-threonine-lysine, whereas in influenza A and B consists of ten hemagglutinin amino acids. A post-translational modification of HEF is the acylation with fatty acids. The fatty acid, stearic acid, was detected to be the prevailing fatty acid attached to HEF, whereas the fatty acid palmitic acid was found in all other membrane proteins. Due to the frequent reassortment of strains, it's monosubtypic and stable. This leads to a new strain that aids the virus in adapting better to its host.


NPAS2

Neuronal PAS domain protein 2 (NPAS2) also known as member of PAS protein 4 (MOP4) is a transcription factor protein that in humans is encoded by the "NPAS2" gene. NPAS2 is paralogous to CLOCK, and both are key proteins involved in the maintenance of circadian rhythms in mammals. In the brain, NPAS2 functions as a generator and maintainer of mammalian circadian rhythms. More specifically, NPAS2 is an activator of transcription and translation of core clock and clock-controlled genes through its role in a negative feedback loop in the suprachiasmatic nucleus (SCN), the brain region responsible for the control of circadian rhythms.
Discovery.
The mammalian and mouse "Npas2" gene was first sequenced and characterized in 1997 Dr. Steven McKnight's lab and published by Yu-Dong Zhou et al. The gene’s cDNAs encoding mouse and human forms of NPAS2 were isolated and sequenced. RNA blotting assays were used to demonstrate the selective presence of the gene in brain and spinal cord tissues of mice. In situ hybridization indicated that the pattern of Npas2 mRNA distribution in mouse brain is broad and complex, and is largely non-overlapping with that of Npas1.
Using Immunohistochemistry of human testis, Ramasamy et al. (2015) found the presence of NPAS2 protein in both germ cells within the tubules of the testes and in the interstitial space of Leydig cells.
Structure.
In humans.
The "Npas2" gene resides on chromosome 2 at the band q13. The gene is 176,679 bases long and contains 25 exons. The predicted 824-amino acid human NPAS2 protein shares 87% sequence identity with mouse "Npas2".
In mice.
The "Npas2" gene has been found to reside on chromosome 1 at 17.98 centimorgans and is 169,505 bases long.
Function.
In the brain.
The NPAS2 protein is a member of the basic helix-loop-helix (bHLH)-PAS transcription factor family and is expressed in the SCN. NPAS2 is a PAS domain-containing protein, which binds other proteins via their own protein-protein (PAS) binding domains. Like its paralogue, CLOCK (another PAS domain-containing protein), the NPAS2 protein can dimerize with the BMAL1 protein and engage in a transcription/translation negative feedback loop (TTFL) to activate transcription of the mammalian "Per" and "Cry" core clock genes. NPAS2 has been shown to form a heterodimer with BMAL1 in both the brain and in cell lines, suggesting its similarity in function to the CLOCK protein in this TTFL.
Compensation is a key feature of TTFLs that regulate circadian rhythms. BMAL1 compensates for CLOCK in that if CLOCK is absent, BMAL1 will upregulate to maintain the mammalian circadian rhythms. NPAS2 has been shown to be analogous to the function of CLOCK in CLOCK-deficient mice. In "Clock" knockout mice, NPAS2 is upregulated to keep the rhythms intact. "Npas2"-mutant mice, which do not express functional NPAS2 protein, still maintain robust circadian rhythms in locomotion. However, like CLOCK-deficient mice in the CLOCK/BMAL1 TTFL, "Npas2"-mutant mice (in the NPAS2/BMAL1 TTFL) still have small defects in their circadian rhythms such as a shortened circadian period and an altered response to changes in the typical light-dark cycle. In addition, "Npas2" knockout mice show sleep disturbances and have decreased expression of "mPer2" in their forebrains. Mice without functional alleles of both "Clock" and "Npas2" became arrhythmic once placed in constant darkness, suggesting that both genes have overlapping roles in maintaining circadian rhythms. In both wild-type and "Clock" knockout mice, "Npas2" expression is observed at the same levels, confirming that "Npas2" plays a role in maintaining these rhythms in the absence of "Clock".
In other tissues.
"Npas2" is expressed everywhere in the periphery of the body. Special focus has been given to its function in liver tissues, and its mRNA is upregulated in "Clock"-mutant mice. However, studies have shown that "Npas2" alone is unable to maintain circadian rhythms in peripheral tissues in the absence of CLOCK protein, unlike in the SCN. One theory to explain this observation is that neurons in the brain are characterized by intercellular coupling and can thus respond to deficiencies in key clock proteins in nearby neurons to maintain rhythms. In peripheral tissues such as the liver and lung, however, the lack of intercellular coupling does not allow for this compensatory mechanism to occur. A second theory as to why NPAS2 can maintain rhythms in CLOCK-deficient SCNs but not in CLOCK-deficient peripheral tissues, is that there exists an additional unknown factor in the SCN that is not present in peripheral tissues.
Non-circadian function.
NPAS2-deficient mice have been shown to have long-term memory deficits, suggesting that the protein may play a key role in the acquisition of such memories. This theory was tested by inserting a reporter gene (lacZ) that resulted in the production of an NPAS2 protein lacking the bHLH domain. These mice were then given several tests, including the cued and contextual fear task, and showed long-term memory deficits in both tasks.
Interactions.
NPAS2 has been shown to interact with:
Clinical significance.
"Npas2" genotypes can be determined through tissue samples from which genomic DNA is extracted and assayed. The assay is performed under PCR conditions and can be used to determine specific mutations and polymorphisms.
Polymorphisms and tumorigenesis.
Mounting evidence suggests that the NPAS2 protein and other circadian genes are involved in tumorigenesis and tumor growth, possibly through their control of cancer-related biologic pathways. A missense polymorphism in NPAS2 (Ala394Thr) has been shown to be associated with risk of human tumors including breast cancer. These findings provide evidence suggesting a possible role for the circadian "Npas2" gene in cancer prognosis. These results have been confirmed in both breast and colorectal cancers.
NPAS2 and mood disorders.
Current research has revealed an association between seasonal affective disorder (SAD) and general mood disorder related to NPAS2, ARNTL, and CLOCK polymorphisms. These genes may influence seasonal variations through metabolic factors such as body weight and appetite.
Associated with a connection to mood disorders, NPAS2 has been found to be involved with dopamine degradation. This was first suggested by the observation that the clock components BMAL1 and NPAS2 transcriptionally activated a luciferase reporter driven by the murine monoamine oxidase A ("Maoa") promoter in a circadian fashion. This suggested that these two clock components (BMAL1 and NPAS2) directly regulated "Maoa" transcription. Subsequent findings discovered positive transcriptional regulation of BMAL1/NPAS2 by PER2. In mice lacking PER2, both "Maoa" mRNA and MAOA protein levels were decreased. Therefore, dopamine degradation was reduced, and dopamine levels in the nucleus accumbens were increased. These findings indicate that degradation of monoamines is regulated by the circadian clock. It is very likely that the described clock-mediated regulation of monoamines is relevant for humans, because single-nucleotide polymorphisms in "Per2, Bmal1", and "Npas2" are associated in an additive fashion with seasonal affective disorder or winter depression.


DNA photolyase

DNA photolyase may refer to:


Sperm-associated antigen 6

Sperm-associated antigen 6 is a protein that in humans is encoded by the "SPAG6" gene.
Function.
The correlation of anti-sperm antibodies with cases of unexplained infertility implicates a role for these antibodies in blocking fertilization. Improved diagnosis and treatment of immunologic infertility, as well as identification of proteins for targeted contraception, are dependent on the identification and characterization of relevant sperm antigens. The protein expressed by this gene is recognized by anti-sperm antibodies from an infertile man. This protein localizes to the tail of permeabilized human sperm and contains eight contiguous armadillo repeats, a motif known to mediate protein-protein interactions. Studies in mice suggest that this protein is involved in sperm flagellar motility and maintenance of the structural integrity of mature sperm. Alternatively spliced variants that encode different protein isoforms have been described but the full-length sequences of only two have been determined.


Mevastatin

Mevastatin (compactin, ML-236B) is a hypolipidemic agent that belongs to the statins class.
It was isolated from the mold "Penicillium citrinum" by Akira Endo in the 1970s, and he identified it as a HMG-CoA reductase inhibitor, i.e., a statin. Mevastatin might be considered the first statin drug; clinical trials on mevastatin were performed in the late 1970s in Japan, but it was never marketed. The first statin drug available to the general public was lovastatin.
Mevastatin has since been derivatized to the compound pravastatin, which is a pharmaceutical used in the lowering of cholesterol and preventing cardiovascular disease.
"In vitro", it has antiproliferative properties.
A British group isolated the same compound from "Penicillium brevicompactum", named it "compactin", and published their results in 1976. The British group mentions antifungal properties with no mention of HMG-CoA reductase inhibition.
High doses inhibit growth and proliferation of melanoma cells.
Biosynthesis.
Biosynthesis of mevastatin is primarily accomplished via a type 1 PKS pathway it proceeds in the PKS pathway as seen in figure 1 until it reaches a hexaketide state where it undergoes a Diels-Alder cyclization. After cyclization it continues via the PKS pathway to a nonaketide after which it is released and undergoes oxidation and dehydration. It is presumed that the oxidations are preformed by a polypeptide that is similar to cytochrome p450 monooxygenase, which is encoded by "mlc"C within the mevastatin gene. Lastly the biosynthesis is completed by the PKS facilitating the addition of a diketide sidechain and a methylation by SAM. Figure 1 shows mevastatin in its acid form but it can also be in the more commonly seen lactone form. This pathway was first observed in "Penicillium cilrinum" and was later discovered that another type of fungus, "Penicillium brevicompaetum" also produced mevastatin via a PKS pathway.
Pharmacology.
Sustained elevations of cholesterol in the blood increase the risk of cardiovascular disease. Mevastatin acts to lowers hepatic production of cholesterol by competitively inhibiting HMG-CoA reductase, the enzyme that catalyzes the rate-limiting step in the cholesterol biosynthesis pathway via the mevalonic acid pathway. When hepatic cholesterol levels are decreased it causes an increased uptake of low density lipoprotein (LDL) cholesterol and reduces cholesterol levels in the circulation. It has also been shown that mevastatin upregulates endothelial nitric oxide synthase (eNOS) in mice, which is essential for maintaining a healthy cardiovascular system.


GDP-4-dehydro-6-deoxy-D-mannose reductase

In enzymology, a GDP-4-dehydro-6-deoxy-D-mannose reductase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are GDP-6-deoxy-D-mannose, NAD+, and NADP+, whereas its 4 products are GDP-4-dehydro-6-deoxy-D-mannose, NADH, NADPH, and H+.
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 GDP-6-deoxy-D-mannose:NAD(P)+ 4-oxidoreductase (D-rhamnose-forming). Other names in common use include GDP-4-keto-6-deoxy-D-mannose reductase [ambiguous], GDP-6-deoxy-D-lyxo-4-hexulose reductase, and Rmd. This enzyme participates in fructose and mannose metabolism.


Apyrase

Apyrase (, "ATP-diphosphatase", adenosine diphosphatase, "ADPase", "ATP diphosphohydrolase") is a calcium-activated plasma membrane-bound enzyme (magnesium can also activate it) () that catalyses the hydrolysis of ATP to yield AMP and inorganic phosphate. Two isoenzymes are found in commercial preparations from "S. tuberosum". One with a higher ratio of substrate selectivity for ATP:ADP (approx 10) and another with no selectivity (ratio 1).
It can also act on ADP and other nucleoside triphosphates and diphosphates with the general reaction being NTP -> NDP + Pi -> NMP + 2Pi. This is the same activity that has been employed in the degradation of unincorporated nucleosides during pyrosequencing.
The salivary apyrases of blood-feeding arthropods are nucleotide hydrolysing enzymes that are implicated in the inhibition of host platelet aggregation through the hydrolysis of extracellular adenosine diphosphate.


4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase

In enzymology, a 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase () is an enzyme that catalyzes the chemical reaction
Hence, this enzyme has one substrate, 4-deoxy-L-threo-5-hexosulose uronate, and one product, 3-deoxy-D-glycero-2,5-hexodiulosonate.
This enzyme belongs to the family of isomerases, specifically those intramolecular oxidoreductases interconverting aldoses and ketoses. The systematic name of this enzyme class is 4-deoxy-L-threo-5-hexosulose-uronate aldose-ketose-isomerase. This enzyme is also called 4-deoxy-L-threo-5-hexulose uronate isomerase. This enzyme participates in pentose and glucuronate interconversions.
Structural studies.
As of late 2007, 3 structures have been solved for this class of enzymes, with PDB accession codes , , and .


BCKDH

BCKDH may refer to:


Nidogen

Nidogens, formerly known as entactins, are a family of sulfated monomeric glycoproteins located in the basal lamina. Two nidogens have been identified in humans: nidogen-1 (NID1) and nidogen-2 (NID2). Remarkably, vertebrates are still capable of stabilizing basement membrane in the absence of either identified nidogen. In contrast, those lacking both nidogen-1 and nidogen-2 typically die prematurely during embryonic development as a result of defects existing in the heart and lungs. Nidogen have been shown to play a crucial role during organogenesis in late embryonic development, particularly in cardiac and lung development. From an evolutionary perspective, nidogens are highly conserved across vertebrates and invertebrates, retaining their ability to bind laminin.
In nematodes, nidogen-1 is necessary for axon guidance, but not for basement membrane assembly.


Bufagin

Bufagin is a toxic steroid C24H34O5 obtained from toad's milk, the poisonous secretion of a skin gland on the back of the neck of a large toad ("Rhinella marina", synonym "Bufo marinus", the cane toad). The toad produces this secretion when it is injured, scared or provoked. Bufagin resembles chemical substances from digitalis in physiological activity and chemical structure.
"Bufagin" also refers to any of several similar substances found as components of the mixture bufotoxin in secretions of other toads, as well as plants and mushrooms.
Chemistry.
Bufagin and bufagins are bufadienolide derivatives. This means they are steroids with a pyran- (delta-)lactone ring attached to ring D (the five-membered one). The difference to digitalis compounds is that the latter have a furan- (gamma-)lactone ring that has one carbon atom and one double bond less.
Effects of bufagins.
Some bufagins have effects similar to poisoning by digitalis, having effects on the cardiac muscle, causing ventricular fibrillation. Some also have local anesthetic action. The analgesic effects have also been proven, by acting as Na+/K+-ATPase inhibitors on the binding sites of the cell membrane. The anti-cancer properties in leukemia and melanoma cells, and the inhibition of the proliferation of prostate cancer cells, have also been investigated in cellular models. Some of the bufagin toxins are used in low doses in traditional Chinese medicine for similar applications as digoxin is used for in Western medicine (i.e. treatment of heart conditions such as atrial fibrillation).
There are at least 86 identified bufadienolides. Examples of bufagins are:
These bufagins, and especially cinobufagin, have given rise to a large number of derivatives evaluated as potential anti-tumor drugs.


KanF

kanF may refer to:


Lysolecithin acyltransferase

Lysolecithin acyltransferase may refer to:


Ring Pop

Ring Pop is a brand of fruit flavored lollipops manufactured by Topps. They are in the form of a wearable plastic ring with a large hard candy "jewel" and come in an assortment of flavors.
History.
Ring Pops were invented in the late 1970s by a man named Frank Richards who was a product engineer at the Topps Company. He wanted to help his daughter in breaking her thumb-sucking habit, so he invented the Ring Pop as a treat to eliminate her childish addiction. This treat is similar to a baby pacifier, where the jewel is made out of a hard candy, and is attached to a plastic disc that clamps around a finger. It was thought to be a brilliant idea and solution to get kids to stop their thumb-sucking habit, while enjoying a sugary and sweet piece of candy instead of sucking on their bland thumb. While the Ring Pop was not the first piece of candy jewelry, it did follow in the footsteps of other jewelry themed candies, such as the candy necklace and candy bracelet, both of which were released in 1958, almost two decades before the invention of the Ring Pop. Candy jewelry was thought to be on the more feminine side, however Topps insured to provide colors that would suit to the male consumers as well, such as blue and green.
Media.
The debut of Ring Pops on television was in the early 1980s, catching many children's attention with its memorable theme song and the famous phrase, "It's a lollipop, without a stick! A ring of flavor you can lick!" The commercial entails many children eating and having fun with Ring Pops, however there is a scene with two children, a boy and a girl, where the little boy is supposedly proposing to the little girl with the Ring Pop. This scene has become a signature staple over the years as one of the most memorable scenes in Ring Pop commercial history.
Variations.
The candy originally was available in two flavors, Cherry and Grape. Over time, Ring Pop flavors have expanded into an array of flavors, such as Blue Raspberry, Strawberry, Watermelon, Lime, and Apple. Ring Pop Sours came into the mix in the 1980s in flavors such as, Sour Green Apple, Sour Cherry Berry, Sour Watermelon, Sour Lemonade, Sour Cherry, and Sour Raspberry Lemonade. Topps has also released Ring Pop "twisted" multi-flavored lollipops, which come in flavors such as, Berry Blast and Citrus Craze, as well as creamy varieties, such as Strawberry Ice Cream. Most recently, in 2013, Topps has come out with Ring Pop Gummies as an alternative to the original hard candy. Special Ring Pops are made for some holidays. Easter Ring Pops use the traditional plastic ring but replace the gem-shaped hard candy with chicks or bunnies.
In 2011, Bazooka issued Ring Pops encrusted with Swarovski Crystal and colored with Maverick Blue to the Dallas Mavericks for their winning of the 2011 NBA Finals.
Topps launched Ring Pop Gummies in 2013, a wearable and edible gummi version of the candy.
Dietary.
Bazooka Candy Brands, a division of Topps Inc., claims Ring Pops to be one of their best selling candies amongst the Orthodox Union because it is one of the few candies that are kosher.


Beta2-adaptin C-terminal domain

The C-terminal domain of Beta2-adaptin is a protein domain is involved in cell trafficking by aiding import and export of substances in and out of the cell.
Function.
This is an adaptor protein which helps the formation of a clathrin coat around a vesicle.
Structure.
This entry represents a subdomain of the appendage (ear) domain of beta-adaptin from AP clathrin adaptor complexes. This domain has a three-layer arrangement, alpha-beta-alpha, with a bifurcated antiparallel beta-sheet. This domain is required for binding to clathrin, and its subsequent polymerisation. Furthermore, a hydrophobic patch present in the domain also binds to a subset of D-phi-F/W motif-containing proteins that are bound by the alpha-adaptin appendage domain (epsin, AP180, eps15).
Cell trafficking.
Proteins synthesized on the ribosome and processed in the endoplasmic reticulum are transported from the Golgi apparatus to the trans-Golgi network (TGN), and from there via small carrier vesicles to their final destination compartment. These vesicles have specific coat proteins (such as clathrin or coatomer) that are important for cargo selection and direction of transport. Clathrin coats contain both clathrin (acts as a scaffold) and adaptor complexes that link clathrin to receptors in coated vesicles. Clathrin-associated protein complexes are believed to interact with the cytoplasmic tails of membrane proteins, leading to their selection and concentration. The two major types of clathrin adaptor complexes are the heterotetrameric adaptor protein (AP) complexes, and the monomeric GGA (Golgi-localising, Gamma-adaptin ear domain homology, ARF-binding proteins) adaptors.
AP (adaptor protein) complexes are found in coated vesicles and clathrin-coated pits. AP complexes connect cargo proteins and lipids to clathrin at vesicle budding sites, as well as binding accessory proteins that regulate coat assembly and disassembly (such as AP180, epsins and auxilin). There are different AP complexes in mammals. AP1 is responsible for the transport of lysosomal hydrolases between the TGN and endosomes. AP2 associates with the plasma membrane and is responsible for endocytosis. AP3 is responsible for protein trafficking to lysosomes and other related organelles. AP4 is less well characterised. AP complexes are heterotetramers composed of two large subunits (adaptins), a medium subunit (mu) and a small subunit (sigma). For example, in AP1 these subunits are gamma-1-adaptin, beta-1-adaptin, mu-1 and sigma-1, while in AP2 they are alpha-adaptin, beta-2-adaptin, mu-2 and sigma-2. Each subunit has a specific function. Adaptins recognise and bind to clathrin through their hinge region (clathrin box), and recruit accessory proteins that modulate AP function through their C-terminal ear (appendage) domains. Mu recognises tyrosine-based sorting signals within the cytoplasmic domains of transmembrane cargo proteins. One function of clathrin and AP2 complex-mediated endocytosis is to regulate the number of GABA(A) receptors available at the cell surface .
More information about these proteins can be found at Protein of the Month: Clathrin .


Dipeptidyl-peptidase II

Dipeptidyl-peptidase II (, "dipeptidyl aminopeptidase II", "dipeptidyl arylamidase II", "carboxytripeptidase", "dipeptidyl peptidase II", "DAP II", "dipeptidyl(amino)peptidase II", "dipeptidylarylamidase") is an enzyme. This enzyme catalyses the following chemical reaction
This lysosomal serine-type peptidase is maximally active at acidic pH.


Peptidoglycan recognition protein

Peptidoglycan recognition proteins (PGRPs) are a group of highly conserved pattern recognition receptors with at least one peptidoglycan recognition domain capable of recognizing the peptidoglycan component of the cell wall of bacteria. They are present in insects, mollusks, echinoderms and chordates. The mechanism of action of PGRPs varies between taxa. In insects, PGRPs kill bacteria indirectly by activating one of four unique effector pathways: prophenoloxidase cascade, Toll pathway, IMD pathway, and induction of phagocytosis. In mammals, PGRPs either kill bacteria directly by interacting with their cell wall or outer membrane, or hydrolyze peptidoglycan. They also modulate inflammation and microbiome and interact with host receptors.
Discovery.
The first PGRP was discovered in 1996 by Masaaki Ashida and coworkers, who purified a 19 kDa protein present in the hemolymph and cuticle of a silkworm ("Bombyx mori"), and named it Peptidoglycan Recognition Protein, because it specifically bound peptidoglycan and activated the prophenoloxidase cascade. In 1998 Håkan Steiner and coworkers, using a differential display screen, identified and cloned a PGRP ortholog in a moth ("Trichoplusia" "ni") and then discovered and cloned mouse and human PGRP orthologs, thus showing that PGRPs are highly conserved from insects to mammals. Also in 1998, Sergei Kiselev and coworkers independently discovered and cloned a protein from a mouse adenocarcinoma with the same sequence as PGRP, which they named Tag7. In 1999 Masanori Ochiai and Masaaki Ashida cloned the silkworm ("B. mori") PGRP.
In 2000, based on the available sequence of the fruit fly ("Drosophila melanogaster") genome, Dan Hultmark and coworkers discovered a family of 12 highly diversified PGRP genes in "Drosophila", which they classified into short (S) and long (L) forms based on the size of their transcripts. By homology searches of available sequences, they also predicted the presence of a long form of human and mouse PGRP (PGRP-L).
In 2001, Roman Dziarski and coworkers discovered and cloned three human PGRPs, named PGRP-L, PGRP-Iα, and PGRP-Iβ (for long and intermediate size transcripts). They established that human genome codes for a family of 4 PGRPs: PGRP-S (short PGRP) and PGRP-L, PGRP-Iα, and PGRP-Iβ. Subsequently, the Human Genome Organization Gene Nomenclature Committee changed the gene symbols of PGRP-S, PGRP-L, PGRP-Iα, and PGRP-Iβ to "PGLYRP1", "PGLYRP2", "PGLYRP3", and "PGLYRP4", respectively, and this nomenclature is currently also used for other mammalian PGRPs. Sergei Kiselev and coworkers also independently cloned mouse PGLYRP2 (TagL). Thereafter, PGRPs have been identified throughout the animal kingdom, although lower metazoa (e.g., the nematode "Caenorhabditis elegans") and plants do not have PGRPs.
In 2003, Byung-Ha Oh and coworkers crystalized PGRP-LB from "Drosophila" and solved its structure.
Types.
Insects generate up to 19 alternatively spliced PGRPs, classified into long (L) and short (S) forms. For instance, the fruit fly ("D. melanogaster") has 13 PGRP genes, whose transcripts are alternatively spliced into 19 proteins, while the mosquito ("Anopheles gambiae") has 7 PGRP genes, with 9 splice variants.
Mammals have up to four PGRPs, all of which are secreted. These are peptidoglycan recognition protein 1 (PGLYRP1), peptidoglycan recognition protein 2 (PGLYRP2), peptidoglycan recognition protein 3 (PGLYRP3) and peptidoglycan recognition protein 4 (PGLYRP4).
Structure.
PGRPs contain at least one C-terminal peptidoglycan recognition domain (PGRP domain), which is about 165 amino acids long. This peptidoglycan-binding type 2 amidase domain is homologous to bacteriophage and bacterial type 2 amidases.
PGRP domain has three peripheral α-helices and several central β-strands that form a peptidoglycan-binding groove on the front face of the molecule, whereas the back of the molecule has a PGRP-specific segment, which is often hydrophobic, diverse among various PGRPs, and not present in bacteriophage amidases.
Invertebrate PGRPs can be small secreted proteins (e.g., PGRP-SB, -SA, -SD, and -LB in "Drosophila"), larger transmembrane proteins (e.g., PGRP-LA, -LC, and -LF in "Drosophila"), or intracellular proteins (e.g., PGRP-LEfl in "Drosophila"). They usually have one C-terminal PGRP domain, with few exceptions, such as "Drosophila" PGRP-LF, which has two PGRP domains.  Mammalian PGRPs are secreted proteins that typically form dimers and contain either one PGRP domain (e.g., human PGLYRP1 and PGLYRP2) or two PGRP domains (e.g., human PGLYRP3 and PGLYRP4).
Functions.
Peptidoglycan binding.
PGRPs bind peptidoglycan, the main component of bacterial cell wall. Peptidoglycan is a polymer of β(1-4)-linked "N"-acetylglucosamine (GlcNAc) and "N"-acetylmuramic acid (MurNAc) cross-linked by short peptides composed of alternating L- and D-amino acids. MurNAc-tripeptide is the minimum fragment of peptidoglycan that binds to PGRPs and MurNAc-tetrtapeptides and MurNAc-pentapeptides bind with higher affinity. Peptidoglycan binding usually induces a change in the structure of PGRP or interaction with another PGRP molecule that locks MurNAc-peptide in the binding grove. Some PGRPs can discriminate between different amino acids present in the peptide part of peptidoglycan, especially between the amino acid in the third position of peptidoglycan peptide, which is usually L-lysine in Gram-positive cocci or meso-diaminopimelic acid ("m"-DAP) in Gram-negative bacteria and Gram-positive bacilli. Some PGRPs can also discriminate between MurNAc and its anhydro form.
Functions in insects.
PGRPs are the main sensors of bacteria in insects and the main components of their antimicrobial defenses. PGRPs activate signaling cascades that induce production of antimicrobial peptides and other immune effectors. Soluble PGRPs (e.g. PGRP-SA and PGRP-SD in "Drosophila") detect L-lysine-containing peptidoglycan and activate a proteolytic cascade that generates an endogenous ligand Spätzle that activates cell-surface Toll-1 receptor. Toll-1 in turn triggers a signal transduction cascade that results in production of antimicrobial peptides primarily active against Gram-positive bacteria and fungi.
Transmembrane PGRPs (e.g., "Drosophila" PGRP-LC) and intracellular PGRPs (e.g., "Drosophila" PGRP-LE) function as receptors – they detect "m"-DAP-containing peptidoglycan and activate IMD (immunodeficiency) signal transduction pathway that induces production of antimicrobial peptides active primarily against Gram-negative bacteria. This activation of IMD pathway also induces production of dual oxidase, which generates antimicrobial reactive oxygen species.
Some insect PGRPs (e.g., "Drosophila" PGRP-SA and -LE, and "B. mori" PGRP-S) activate the prophenoloxidase cascade, which results in the formation of melanin, reactive oxygen species, and other antimicrobial compounds.
Several small insect PGRPs (e.g., "Drosophila" PGRP-SB, -SC, and -LB) are peptidoglycan hydrolases ("N"-acetylmuramoyl-L-alanine amidases) that hydrolyzes the amide bond between the MurNAc and L-Ala (the first amino acid in the stem peptide). These amidases act as peptidoglycan scavengers because they render the resulting peptidoglycan fragments unable to bind to PGRP. They abolish cell-activating capacity of peptidoglycan and limit systemic uptake of peptidoglycan from the bacteria-laden intestinal tract and down-regulate or prevent over-activation of host defense pathways. Some of these amidases are also directly bactericidal, which further defends the host against infections and helps to control the numbers of commensal bacteria.
Some other insect PGRPs (e.g., "Drosophila" PGRP-LF) do not bind peptidoglycan and lack intracellular signaling domain – they complex with PGRP-LC and function to down-regulate activation of the IMD pathway.
Functions in other invertebrates.
PGRPs are present and constitutively expressed or induced by bacteria in most invertebrates, including worms, snails, oysters, scallops, squid, and starfish. These PGRPs are confirmed or predicted amidases and some have antibacterial activity. They likely defend the hosts against infections or regulate colonization by certain commensal bacteria, such as "Vibrio fischeri" in the light organ of Hawaiian bobtail squid, "Euprymna scolopes".
Expression and functions in lower vertebrates.
Early fish-like chordates, amphioxi (lancelets), have extensive innate immune system (but no adaptive immunity) and have multiple "PGRP" genes – e.g., 18 "PGRP" genes in the Florida lancelet ("Branchiostoma floridae"), all of which are predicted peptidoglycan-hydrolyzing amidases and at least one is bactericidal.
Fish, such as zebrafish ("Danio rerio"), typically have 4 "PGRP" genes, but they are not all orthologous to mammalian "PGLYRPs" and different species may have multiple "PGRP" splice variants. They are constitutively expressed in many tissues of adult fish, such as liver, gills, intestine, pancreas, spleen, and skin, and bacteria can increase their expression. PGRPs are also highly expressed in developing oocytes and in eggs (e.g., zebrafish PGLYRP2 and PGLYRP5). These PGRPs have both peptidoglycan-hydrolyzing amidase activity and are directly bactericidal to both Gram-positive and Gram-negative bacteria and protect eggs and developing embryos from bacterial infections. They may also regulate several signaling pathways.
Amphibian PGRPs are also proven or predicted amidases and likely have similar functions to fish PGRPs.
Expression in mammals.
All four mammalian PGRPs are secreted proteins.
PGLYRP1 (peptidoglycan recognition protein 1) has the highest level of expression of all mammalian PGRPs. PGLYRP1 is highly constitutively expressed in the bone marrow and in the granules of neutrophils and eosinophils, and also in activated macrophages, lactating mammary gland, and intestinal Peyer's patches’ microfold (M) cells, and to a much lesser extent in epithelial cells in the eye, mouth, and respiratory and intestinal tracts.
PGLYRP2 (peptidoglycan recognition protein 2) is constitutively expressed in the liver, from where it is secreted into the blood. Liver PGLYRP2 and earlier identified serum "N"-acetylmuramoyl-L-alanine amidase are the same protein encoded by the "PGLYRP2" gene. Bacteria and cytokines induce low level of PGLYRP2 expression in the skin and gastrointestinal epithelial cells, intestinal intraepithelial T lymphocytes, dendritic cells, NK (natural killer) cells, and inflammatory macrophages. Some mammals, e.g. pigs, express multiple splice forms of PGLYRP2 with differential expression.
PGLYRP3 (peptidoglycan recognition protein 3) and PGLYRP4 (peptidoglycan recognition protein 4) are constitutively expressed in the skin, in the eye, and in mucous membranes in the tongue, throat, and esophagus, and at a much lower level in the remaining parts of the intestinal tract. PGLYRP4 is also expressed in the salivary glands and mucus-secreting glands in the throat. Bacteria and their products increase expression of PGLYRP3 and PGLYRP4 in keratinocytes and oral epithelial cells. When expressed in the same cells, PGLYRP3 and PGLYRP4 form disulfide-linked heterodimers.
Mouse PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4 are also differentially expressed in the developing brain and this expression is influenced by the intestinal microbiome. Expression of PGLYRP1 is also induced in rat brain by sleep deprivation and in mouse brain by ischemia.
Functions in mammals.
Human PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal for both Gram-positive and Gram-negative bacteria. Mouse and bovine PGLYRP1 also have antibacterial activity, and bovine PGLYRP1 has also antifungal activity. These human PGRPs kill bacteria by simultaneously inducing three synergistic stress responses: oxidative stress, thiol stress, and metal stress. Bacterial killing by these PGRPs does not involve cell membrane permeabilization, cell wall hydrolysis, or osmotic shock, but is synergistic with lysozyme and antibacterial peptides.
Human, mouse, and porcine PGLYRP2 are enzymes, "N"-acetylmuramoyl-L-alanine amidases, that hydrolyze the amide bond between the MurNAc and L-alanine, the first amino acid in the stem peptide in bacterial cell wall peptidoglycan. The minimal peptidoglycan fragment hydrolyzed by PGLYRP2 is MurNAc-tripeptide. Hydrolysis of peptidoglycan by PGLYRP2 diminishes its pro-inflammatory activity.
Unlike invertebrate and lower vertebrate PGRPs, mammalian PGRPs have only limited role in defense against infections. Intranasal application of PGLYRP3 or PGLYRP4 in mice protects from intranasal lung infection with "Staphylococcus aureus" and "Escherichia" "coli", and intravenous administration of PGLYRP1 protects mice from systemic "Listeria monocytogenes" infection. Also, "PGLYRP1"-deficient mice are more sensitive to systemic infections with non-pathogenic bacteria ("Micrococcus luteus" and "Bacillus" "subtilis") and to "Pseudomonas aeruginosa"-induced keratitis, but not to systemic infections with pathogenic bacteria ("S. aureus" and "E. coli"). "PGLYRP2"-deficient mice are more sensitive to "P. aeruginosa"-induced keratitis and "Streptococcus" "pneumoniae"-induced pneumonia and sepsis, and "PGLYRP4"-deficient mice are more sensitive to "S. pneumoniae"-induced pneumonia.
Mouse PGRPs play a role in maintaining healthy microbiome, as "PGLYRP1"-, "PGLYRP2"-, "PGLYRP3"-, and "PGLYRP4"-deficient mice have significant changes in the composition of their intestinal microbiomes and "PGLYRP1"-deficient mice also have changes in their lung microbiome.
Mouse PGRPs also play a role in maintaining anti- and pro-inflammatory homeostasis in the intestine, skin, lungs, joints, and brain. All four PGLYRPs protect mice from dextran sodium sulfate (DSS)-induced colitis and the effect of PGLYRP2 and PGLYRP3 on the intestinal microbiome is responsible for this protection. PGLYRP3 is anti-inflammatory in intestinal epithelial cells. PGLYRP4 has anti-inflammatory effect in a mouse model of "S. pneumoniae" pneumonia and sepsis, which also depends on the PGLYRP4-controlled microbiome.
PGLYRP3 and PGLYRP4 are anti-inflammatory and protect mice from atopic dermatitis and PGLYRP4 also protects mice from "Bordetella pertussis"-induced airway inflammation. PGLYRP2 is anti-inflammatory and protects mice from experimentally-induced psoriasis-like inflammation and "Salmonella enterica"-induced intestinal inflammation. But some PGRPs have opposite effects: PGLYRP2 has also pro-inflammatory effects, as it promotes the development of experimental arthritis, bacterially-induced keratitis, and inflammation in "S. pneumoniae" lung infection in mice. PGLYRP1 is pro-inflammatory and promotes experimentally-induced asthma and skin inflammation in mice, and this pro-inflammatory effect on asthma depends on the PGLYRP1-regulated intestinal microbiome.
PGLYRP1 also promotes wound healing in experimentally-induced keratitis in mice [64], whereas PGLYRP2 regulates motor activity and anxiety-dependent behavior in mice.
Some mammalian PGRPs can also function as host receptor agonists or antagonists. Human PGLYRP1 complexed with peptidoglycan or multimerized binds to and stimulates TREM-1 (triggering receptor expressed on myeloid cells-1), a receptor present on neutrophils, monocytes and macrophages that induces production of pro-inflammatory cytokines.
Human and mouse PGLYRP1 (Tag7) bind heat shock protein 70 (Hsp70) in solution and PGLYRP1-Hsp70 complexes are also secreted by cytotoxic lymphocytes, and these complexes are cytotoxic for tumor cells. This cytotoxicity is antagonized by metastasin (S100A4) and heat shock-binding protein HspBP1. PGLYRP1-Hsp70 complexes bind to the TNFR1 (tumor necrosis factor receptor-1, which is a death receptor) and induce a cytotoxic effect via apoptosis and necroptosis. This cytotoxicity is associated with permeabilization of lysosomes and mitochondria. By contrast, free PGLYRP1 acts as a TNFR1 antagonist by binding to TNFR1 and inhibiting its activation by PGLYRP1-Hsp70 complexes. A peptide from human PGLYRP1 (amino acids 163-175) also inhibits the cytotoxic effects of TNF-α and PGLYRP1-Hsp70 complexes.
Medical relevance.
Genetic "PGLYRP" variants or changed expression of PGRPs are associated with several diseases. Patients with inflammatory bowel disease (IBD), which includes Crohn’s disease and ulcerative colitis, have significantly more frequent missense variants in all four "PGLYRP" genes than healthy controls. These results suggest that PGRPs protect humans from these inflammatory diseases, and that mutations in "PGLYRP" genes are among the genetic factors predisposing to these diseases. "PGLYRP1" variants are also associated with increased fetal hemoglobin in sickle cell disease, "PGLYRP2" variants are associated with esophageal squamous cell carcinoma, "PGLYRP2", "PGLYRP3", and "PGLYRP4" variants are associated with Parkinson’s disease, "PGLYRP3" and "PGLYRP4" variants are associated with psoriasis and composition of airway microbiome, and PGLYRP4 variants are associated with ovarian cancer.
Several diseases are associated with increased expression of PGLYRP1, including: atherosclerosis, myocardial infarction, heart failure, sepsis, pulmonary fibrosis, asthma, chronic kidney disease, rheumatoid arthritis, gingival inflammation, osteoarthritis, cardiovascular events and death in kidney transplant patients, alopecia, type I diabetes, infectious complications in hemodialysis, and thrombosis, consistent with pro-inflammatory effects of PGLYRP1. Lower expression of PGLYRP1 was found in endometriosis.
Decreased expression of PGLYRP2 is associated with HIV-associated tuberculosis, Lyme disease, hepatocellular carcinoma, and myocardial infarction.
Applications.
A silkworm larvae plasma (SLP) test to detect peptidoglycan, based on activation of the prophenoloxidase cascade by PGRP in the hemolymph of the silkworm, "Bombyx mori", is available.


H.C. Prinsen Geerligs

Hendrik Coenraad Prinsen Geerligs (24 November 1864 – 31 July 1953) was a Dutch chemist and sugar technologist active in Java in the early part of the 20th century.
Biography.
Prinsen Geerligs was born in Haarlem, Netherlands on 24 November 1864. He was the son of a schoolmaster and did his primary and high schooling in Haarlem and later at the public in Amsterdam he wrote the final examinations there in 1883. He then studied chemistry at the University of Amsterdam. He worked in the chemical/technical office of the firm Wijnhoff en van Gulpen until 1891. After which he went to Java to work as an assistant chemist at the Kagok Sugar Research Station.
He wrote a number of books on sugar manufacturing.
He was a member of the Royal Dutch Academy of Science and their correspondent for the Natural Sciences section from 9 May 1899 to 1 October 1909. He was elected a member of the in 1907.


2-aminohexanoate transaminase

In enzymology, a 2-aminohexanoate transaminase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are L-2-aminohexanoate and 2-oxoglutarate, whereas its two products are 2-oxohexanoate and L-glutamate.
This enzyme belongs to the family of transferases, specifically the transaminases, which transfer nitrogenous groups. The systematic name of this enzyme class is L-2-aminohexanoate:2-oxoglutarate aminotransferase. Other names in common use include norleucine transaminase, norleucine (leucine) aminotransferase, and leucine L-norleucine: 2-oxoglutarate aminotransferase. It employs one cofactor, pyridoxal phosphate.


(2Z,6Z)-farnesyl diphosphate lyase

(2Z,6Z)-farnesyl diphosphate lyase may refer to:


Ectonucleotidase

Ectonucleotidases consist of families of nucleotide metabolizing enzymes that are expressed on the plasma membrane and have externally oriented active sites. These enzymes metabolize nucleotides to nucleosides. The contribution of ectonucleotidases in the modulation of purinergic signaling depends on the availability and preference of substrates and on cell and tissue distribution.
Classification.
Subfamilies of ectonucleotidases include: CD39/NTPDases (ecto-nucleotide triphosphate diphosphohydrolases), Nucleotide pyrophosphatase/phosphodiesterase (NPP)-type ecto-phosphodiesterases, alkaline phosphatases and ecto-5’-nucleotidases/CD73.
Function.
Ectonucleotidases produce key molecules for purine salvage and consequent replenishment of ATP stores within multiple cell types. Dephosphorylated nucleoside derivatives interact with membrane transporters to enable intracellular uptake. Ectonucleotidases modulate P2 purinergic signaling, and P1 receptors. In addition, ectonucleotidases generate extracellular adenosine, which abrogates nucleotide-mediated effects and activates adenosine receptors, often with opposing (patho-) physiological effects.
Adenosine generation.
The first step in the production of adenosine involves the conversion of ATP/ADP to AMP. It is carried out by ENTPD1, also known as CD39. The second step involves the conversion of AMP to adenosine. It is carried out by NT5E, also known as CD73.


Alg2 mannosyltransferase

Alg2 mannosyltransferase may refer to:


RABEPK

Rab9 effector protein with Kelch motifs also known as p40 is a protein that in humans is encoded by the "RABEPK" gene.
Membrane-associated p40, in together with RAB9A, facilitates the transport of the mannose 6-phosphate receptor (MPR) from endosomes to the trans-Golgi network.
Interactions.
RABEPK has been shown to interact with RAB9A and FYVE finger-containing phosphoinositide kinase.


Chondroitinase

Chondroitinase may refer to:


Exoenzyme

An exoenzyme, or extracellular enzyme, is an enzyme that is secreted by a cell and functions outside that cell. Exoenzymes are produced by both prokaryotic and eukaryotic cells and have been shown to be a crucial component of many biological processes. Most often these enzymes are involved in the breakdown of larger macromolecules. The breakdown of these larger macromolecules is critical for allowing their constituents to pass through the cell membrane and enter into the cell. For humans and other complex organisms, this process is best characterized by the digestive system which breaks down solid food via exoenzymes. The small molecules, generated by the exoenzyme activity, enter into cells and are utilized for various cellular functions. Bacteria and fungi also produce exoenzymes to digest nutrients in their environment, and these organisms can be used to conduct laboratory assays to identify the presence and function of such exoenzymes. Some pathogenic species also use exoenzymes as virulence factors to assist in the spread of these disease-causing microorganisms. In addition to the integral roles in biological systems, different classes of microbial exoenzymes have been used by humans since pre-historic times for such diverse purposes as food production, biofuels, textile production and in the paper industry. Another important role that microbial exoenzymes serve is in the natural ecology and bioremediation of terrestrial and marine environments.
History.
Very limited information is available about the original discovery of exoenzymes. According to Merriam-Webster dictionary, the term "exoenzyme" was first recognized in the English language in 1908. The book "Intracellular Enzymes: A Course of Lectures Given in the Physiological," by Horace Vernon is thought to be the first publication using this word in that year. Based on the book, it can be assumed that the first known exoenzymes were pepsin and trypsin, as both are mentioned by Vernon to have been discovered by scientists Briike and Kiihne before 1908.
Function.
In bacteria and fungi, exoenzymes play an integral role in allowing the organisms to effectively interact with their environment. Many bacteria use digestive enzymes to break down nutrients in their surroundings. Once digested, these nutrients enter the bacterium, where they are used to power cellular pathways with help from endoenzymes.
Many exoenzymes are also used as virulence factors. Pathogens, both bacterial and fungal, can use exoenzymes as a primary mechanism with which to cause disease. The metabolic activity of the exoenzymes allows the bacterium to invade host organisms by breaking down the host cells' defensive outer layers or by necrotizing body tissues of larger organisms. Many gram-negative bacteria have injectisomes, or flagella-like projections, to directly deliver the virulent exoenzyme into the host cell using a type three secretion system. With either process, pathogens can attack the host cell's structure and function, as well as its nucleic DNA.
In eukaryotic cells, exoenzymes are manufactured like any other enzyme via protein synthesis, and are transported via the secretory pathway. After moving through the rough endoplasmic reticulum, they are processed through the Golgi apparatus, where they are packaged in vesicles and released out of the cell. In humans, a majority of such exoenzymes can be found in the digestive system and are used for metabolic breakdown of macronutrients via hydrolysis. Breakdown of these nutrients allows for their incorporation into other metabolic pathways.
Examples of exoenzymes as virulence factors.
Necrotizing enzymes.
Necrotizing enzymes destroy cells and tissue. One of the best known examples is an exoenzyme produced by Streptococcus pyogenes that causes necrotizing fasciitis in humans.
Coagulase.
By binding to prothrombin, coagulase facilitates clotting in a cell by ultimately converting fibrinogen to fibrin. Bacteria such as Staphylococcus aureus use the enzyme to form a layer of fibrin around their cell to protect against host defense mechanisms.
Kinases.
The opposite of coagulase, kinases can dissolve clots. S. aureus can also produce staphylokinase, allowing them to dissolve the clots they form, to rapidly diffuse into the host at the correct time.
Hyaluronidase.
Similar to collagenase, hyaluronidase enables a pathogen to penetrate deep into tissues. Bacteria such as Clostridium do so by using the enzyme to dissolve collagen and hyaluronic acid, the protein and saccharides, respectively, that hold tissues together.
Hemolysins.
Hemolysins target erythrocytes, a.k.a. red blood cells. Attacking and lysing these cells harms the host organism, and provides the microorganism, such as the fungus Candida albicans, with a source of iron from the lysed hemoglobin. Organisms can either by alpha-hemolytic, beta-hemolytic, or gamma-hemolytic (non-hemolytic).
Examples of digestive exoenzymes.
Amylases.
Amylases are a group of extracellular enzymes (glycoside hydrolases) that catalyze the hydrolysis of starch into maltose. These enzymes are grouped into three classes based on their amino acid sequences, mechanism of reaction, method of catalysis and their structure. The different classes of amylases are α-amylases, β-amylases, and glucoamylases. The α-amylases hydrolyze starch by randomly cleaving the 1,4-a-D-glucosidic linkages between glucose units, β-amylases cleave non-reducing chain ends of components of starch such as amylose, and glucoamylases hydrolyze glucose molecules from the ends of amylose and amylopectin. Amylases are critically important extracellular enzymes and are found in plants, animals, and microorganisms. In humans, amylases are secreted by the pancreas and salivary glands, with both sources of the enzyme required for complete starch hydrolysis.
Lipoprotein lipase.
Lipoprotein lipase (LPL) is a type of digestive enzyme that helps regulate the uptake of triacylglycerols from chylomicrons and other low-density lipoproteins from fatty tissues in the body. The exoenzymatic function allows it to break down the triacylglycerol into two free fatty acids and one molecule of monoacylglycerol. LPL can be found in endothelial cells in fatty tissues, such as adipose, cardiac, and muscle. Lipoprotein lipase is downregulated by high levels of insulin, and upregulated by high levels of glucagon and adrenaline.
Pectinase.
Pectinases, also called pectolytic enzymes, are a class of exoenzymes that are involved in the breakdown of pectic substances, most notably pectin. Pectinases can be classified into two different groups based on their action against the galacturonan backbone of pectin: de-esterifying and depolymerizing. These exoenzymes can be found in both plants and microbial organisms including fungi and bacteria. Pectinases are most often used to break down the pectic elements found in plants and plant-derived products.
Pepsin.
Discovered in 1836, pepsin was one of the first enzymes to be classified as an exoenzyme. The enzyme is first made in the inactive form, pepsinogen by chief cells in the lining of the stomach. With an impulse from the vagus nerve, pepsinogen is secreted into the stomach, where it mixes with hydrochloric acid to form pepsin. Once active, pepsin works to break down proteins in foods such as dairy, meat, and eggs. Pepsin works best at the pH of gastric acid, 1.5 to 2.5, and is deactivated when the acid is neutralized to a pH of 7.
Trypsin.
Also one of the first exoenzymes to be discovered, trypsin was named in 1876, forty years after pepsin. This enzyme is responsible for the breakdown of large globular proteins and its activity is specific to cleaving the C-terminal sides of arginine and lysine amino acid residues. It is the derivative of trypsinogen, an inactive precursor that is produced in the pancreas. When secreted into the small intestine, it mixes with enterokinase to form active trypsin. Due to its role in the small intestine, trypsin works at an optimal pH of 8.0.
Bacterial assays.
The production of a particular digestive exoenzyme by a bacterial cell can be assessed using plate assays. Bacteria are streaked across the agar, and are left to incubate. The release of the enzyme into the surroundings of the cell cause the breakdown of the macromolecule on the plate. If a reaction does not occur, this means that the bacteria does not create an exoenzyme capable of interacting with the surroundings. If a reaction does occur, it becomes clear that the bacteria does possess an exoenzyme, and which macromolecule is hydrolyzed determines its identity.
Amylase.
Amylase breaks down carbohydrates into mono- and disaccharides, so a starch agar must be used for this assay. Once the bacteria is streaked on the agar, the plate is flooded with iodine. Since iodine binds to starch but not its digested by-products, a clear area will appear where the amylase reaction has occurred. Bacillus subtilis is a bacterium that results in a positive assay as shown in the picture.
Lipase.
Lipase assays are done using a lipid agar with a spirit blue dye. If the bacteria has lipase, a clear streak will form in the agar, and the dye will fill the gap, creating a dark blue halo around the cleared area. Staphylococcus epidermis results in a positive lipase assay.
Biotechnological and industrial applications.
Microbiological sources of exoenzymes including amylases, proteases, pectinases, lipases, xylanases, cellulases among others are used for a wide range of biotechnological and industrial uses including biofuel generation, food production, paper manufacturing, detergents and textile production. Optimizing the production of biofuels has been a focus of researchers in recent years and is centered around the use of microorganisms to convert biomass into ethanol. The enzymes that are of particular interest in ethanol production are cellobiohydrolase which solubilizes crystalline cellulose and xylanase that hydrolyzes xylan into xylose. One model of biofuel production is the use of a mixed population of bacterial strains or a consortium that work to facilitate the breakdown of cellulose materials into ethanol by secreting exoenzymes such as cellulases and laccases. In addition to the important role it plays in biofuel production, xylanase is utilized in a number of other industrial and biotechnology applications due to its ability to hydrolyze cellulose and hemicellulose. These applications include the breakdown of agricultural and forestry wastes, working as a feed additive to facilitate greater nutrient uptake by livestock, and as an ingredient in bread making to improve the rise and texture of the bread.
Lipases are one of the most used exoenzymes in biotechnology and industrial applications. Lipases make ideal enzymes for these applications because they are highly selective in their activity, they are readily produced and secreted by bacteria and fungi, their crystal structure is well characterized, they do not require cofactors for their enzymatic activity, and they do not catalyze side reactions. The range of uses of lipases encompasses production of biopolymers, generation of cosmetics, use as a herbicide, and as an effective solvent. However, perhaps the most well known use of lipases in this field is its use in the production of biodiesel fuel. In this role, lipases are used to convert vegetable oil to methyl- and other short-chain alcohol esters by a single transesterification reaction.
Cellulases, hemicellulases and pectinases are different exoenzymes that are involved in a wide variety of biotechnological and industrial applications. In the food industry these exoenzymes are used in the production of fruit juices, fruit nectars, fruit purees and in the extraction of olive oil among many others. The role these enzymes play in these food applications is to partially breakdown the plant cell walls and pectin. In addition to the role they play in food production, cellulases are used in the textile industry to remove excess dye from denim, soften cotton fabrics, and restore the color brightness of cotton fabrics. Cellulases and hemicellulases (including xylanases) are also used in the paper and pulp industry to de-ink recycled fibers, modify coarse mechanical pulp, and for the partial or complete hydrolysis of pulp fibers. Cellulases and hemicellulases are used in these industrial applications due to their ability to hydrolyze the cellulose and hemicellulose components found in these materials.
Bioremediation applications.
Bioremediation is a process in which pollutants or contaminants in the environment are removed through the use of biological organisms or their products. The removal of these often hazardous pollutants is mostly carried out by naturally occurring or purposely introduced microorganisms that are capable of breaking down or absorbing the desired pollutant. The types of pollutants that are often the targets of bioremediation strategies are petroleum products (including oil and solvents) and pesticides. In addition to the microorganisms ability to digest and absorb the pollutants, their secreted exoenzymes play an important role in many bioremediation strategies.
Fungi have been shown to be viable organisms to conduct bioremediation and have been used to aid in the decontamination of a number of pollutants including polycyclic aromatic hydrocarbons (PAHs), pesticides, synthetic dyes, chlorophenols, explosives, crude oil, and many others. While fungi can breakdown many of these contaminants intracellularly, they also secrete numerous oxidative exoenzymes that work extracellularly. One critical aspect of fungi in regards to bioremediation is that they secrete these oxidative exoenzymes from their ever elongating hyphal tips. Laccases are an important oxidative enzyme that fungi secrete and use oxygen to oxidize many pollutants. Some of the pollutants that laccases have been used to treat include dye-containing effluents from the textile industry, wastewater pollutants (chlorophenols, PAHs, etc.), and sulfur-containing compounds from coal processing.
Bacteria are also a viable source of exoenzymes capable of facilitating the bioremediation of the environment. There are many examples of the use of bacteria for this purpose and their exoenzymes encompass many different classes of bacterial enzymes. Of particular interest in this field are bacterial hydrolases as they have an intrinsic low substrate specificity and can be used for numerous pollutants including solid wastes. Plastic wastes including polyurethanes are particularly hard to degrade, but an exoenzyme has been identified in a Gram-negative bacterium, "Comamonas acidovorans", that was capable of degrading polyurethane waste in the environment. Cell-free use of microbial exoenzymes as agents of bioremediation is also possible although their activity is often not as robust and introducing the enzymes into certain environments such as soil has been challenging. In addition to terrestrial based microorganisms, marine based bacteria and their exoenzymes show potential as candidates in the field of bioremediation. Marine based bacteria have been utilized in the removal of heavy metals, petroleum/diesel degradation and in the removal of polyaromatic hydrocarbons among others.


Lignin-modifying enzyme

Lignin-modifying enzymes (LMEs) are various types of enzymes produced by fungi and bacteria that catalyze the breakdown of lignin, a biopolymer commonly found in the cell walls of plants. The terms ligninases and lignases are older names for the same class, but the name "lignin-modifying enzymes" is now preferred, given that these enzymes are not hydrolytic but rather oxidative (electron withdrawing) by their enzymatic mechanisms. LMEs include peroxidases, such as lignin peroxidase (), manganese peroxidase (), versatile peroxidase (), and many phenoloxidases of the laccase type.
LMEs have been known to be produced by many species of white rot basidiomycetous fungi, including: "Phanerochaete chrysosporium", "Ceriporiopsis subvermispora", "Trametes versicolor", "Phlebia radiata", "Pleurotus ostreatus" and "Pleurotus eryngii".
LMEs are produced not only by wood-white rotting fungi but also by litter-decomposing basidiomycetous fungi such as "Agaricus bisporus" (common button mushroom), and many "Coprinus" and "Agrocybe" species. The brown-rot fungi, which are able to colonize wood by degrading cellulose, are only able to partially degrade lignin.
Some bacteria also produce LMEs, although fungal LMEs are more efficient in lignin degradation. Fungi are thought to be the most substantial contributors to lignin degradation in natural systems.
LMEs and cellulases are crucial to ecologic cycles (for example, growth/death/decay/regrowth, the carbon cycle, and soil health) because they allow plant tissue to be decomposed quickly, releasing the matter therein for reuse by new generations of life. LMEs are also crucial to a number of different industries.
Industry Application.
Lignin-modifying enzymes have been actively used in the paper and pulp industry for the last decade. They were used in the industry shortly after they were discovered to have both detoxifying and decolorizing properties; properties that the pulp industry spends over $100 million USD annually on to pursue. Although these enzymes have been applied to industry for the last ten years, optimal and robust fermentative processes have not been established. There is an area of active research as scientists believe that the lack of optimal conditions for these enzymes are limiting industrial exploitation.
Lignin-modifying enzymes benefit industry as they can break down lignin; a common waste product of the paper and pulp industry. These enzymes have been used in the refinement of poplar as lignin inhibits the enzymatic hydrolysis of treated poplar and Lignin-modifying enzymes can efficiently degrade the lignin thus fixing this problem.
Another use of lignin modifying enzymes is the optimization of plant biomass use. Historically, only a small fraction of plant biomass use could actually be extracted from pulp sources leaving the majority of the plants as waste products. Due to Lignin, the plant waste is relatively inert towards degradation and causes the large accumulation of waste products. LMEs can effectively break it down into other aromatic compounds.
LMEs were initially used for the bleaching of waste effluent. Now there are several patented processes that make use of these enzymes for pulp bleaching, many of which are still under development.
The environmental industry has interest in using LMEs for the degradation of xenobiotic compounds. There is active research into the detoxification of herbicides by LMEs. Trametes versicolor was shown to effectively degraded Glyphosate in vitro.
Bacterial lignin-modifying enzymes.
Although much research has been done to understand fungal LMEs, only recently has more focus been placed on characterizing these enzymes in bacteria. The main LMEs in both fungi and bacteria are peroxidases and laccases.
Although bacteria lack homologs to the most common fungal peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), many produce dye decolourizing peroxidases (DyP-type peroxidases). Bacteria from a variety of classes express DyP peroxidases, including Gammaproteobacteria, Firmicutes, and Actinobacteria. Peroxidases depolymerize lignin by oxidation using hydrogen peroxide. Fungal peroxidases have higher oxidizing power than bacterial DyP-type peroxidases studied so far, and are able to degrade more complex lignin structures. DyP-type peroxidases have been found to work on a large range of substrates, including synthetic dyes, monophenolic compounds, lignin-derived compounds, and alcohols.
Laccases, which are multicopper oxidases, are another class of enzymes found in both bacteria and fungi which have significant lignin-degrading properties. Laccases degrade lignin by oxidation using oxygen. Laccases are also widely distributed among bacterial species, including "Bacillus subtilis", "Caulobacter crescentus", "Escherichia coli" and "Mycobacterium tuberculosum". Like DyP-type peroxidases, bacterial laccases have a wide substrate range.
There is interest in using bacterial laccases and DyP peroxidases for industry applications, biotechnology and bioremediation because of the greater ease of manipulation of bacterial genomes and gene expression compared to fungi. The wide range of substrates for these types of enzymes also increases the range of processes they may be used in. These processes include pulp processing, textile dye modification, decontamination of waste water and production of pharmaceutical building blocks. Furthermore, bacterial laccases function at higher temperatures, alkalinity, and salt concentrations than fungal laccases, making them more suitable for industrial use.
Both intracellular and extracellular bacterial DyP-type peroxidases and laccases have been identified, suggesting that some are used as intracellular enzymes while others are secreted to degrade compounds in the environment. However, their roles in bacterial physiology and their natural physiological substrates have yet to be detailed.


2,6-dioxo-6-phenylhexa-3-enoate hydrolase

In enzymology, a 2,6-dioxo-6-phenylhexa-3-enoate hydrolase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are 2,6-dioxo-6-phenylhexa-3-enoate and H2O, whereas its two products are benzoate and 2-oxopent-4-enoate.
This enzyme belongs to the family of hydrolases, specifically those acting on carbon-carbon bonds in ketonic substances. The systematic name of this enzyme class is 2,6-dioxo-6-phenylhexa-3-enoate benzoylhydrolase. This enzyme is also called HOHPDA hydrolase. This enzyme participates in biphenyl degradation and fluorene degradation.
Structural studies.
As of late 2007, 3 structures have been solved for this class of enzymes, with PDB accession codes , , and .


Galactosidases

Galactosidases are enzymes (glycoside hydrolases) that catalyze the hydrolysis of galactosides into monosaccharides.
Galactosides can be classified as either alpha or beta. If the galactoside is classified as an alpha-galactoside, the enzyme is called alpha-galactosidase, and is responsible for catalyzing the hydrolysis of substrates that contain α-galactosidic residues, such as glycosphingolipids or glycoproteins. On the other hand, if it is a beta-galactoside, it is called beta-galactosidase, and is responsible for breaking down the disaccharide lactose into its monosaccharide components, glucose and galactose Both varieties of galactosidase are categorized under the EC number 3.2.1.
Two recombinant forms of alpha-galactosidase are called agalsidase alfa (INN) and agalsidase beta (INN). Lack of alpha-galactosidase activity in leukocytes has been linked to Fabry Disease.
Galactosidases have a variety of uses, including the production of prebiotics, the biosynthesis of transgalactosylated products, and the removal of lactose.
B-galactosidase forms the basis of lac z operon in bacteria which can be used to control gene expression.
Uses.
B-galactosidase can be used to track the efficiency of bacterial transformation with a recombinant plasmid in a process called Blue/White Color Screening. B-galactosidase is made up of 4 identical polypeptide chains, i.e. it has 4 identical subunits. When B-galactosidase is separated into 2 fragments, it has the unique property of regaining enzymatic activity upon the rejoining of the inactive fragments. In the process called alpha-complementation, one of the fragments (omega) is encoded by a part of a gene of the lac operon that is found in the chromosome of the bacteria, while the other fragment (alpha) is encoded by the other part of the gene that is found in the plasmid. it is only when both parts of the gene are being expressed that both the omega and alpha fragments are produced. When both fragments are present they would come together to restore the activity of B-galactosidase. However, by placing the target gene within the locus responsible for encoding the alpha fragment, one can track the presence of the desired gene in the plasmid. When the target gene is present, the alpha-fragment gene would be inactive and the alpha fragment won't be produced. In that case B-galactosidase will not be active. When the target gene is not found in the vector, the alpha fragment gene would be active, producing the alpha fragment and allowing for B-galactosidase to gain its activity. To trace the activity of B-galactosidase a colorless analog of lactose is used, X-gal. The hydrolysis of X-gal by B-galactosidase produces galactose, a blue colored compound. Therefore, when the bacteria is transformed with the recombinant plasmid B-galactosidase is inactive and the colonies appear white, but when bacteria are transformed with the original plasmid, lacking the target gene, B-galactosidase is active and the colonies appear blue.


Fatty acid-binding protein

The fatty-acid-binding proteins (FABPs) are a family of transport proteins for fatty acids and other lipophilic substances such as eicosanoids and retinoids. These proteins are thought to facilitate the transfer of fatty acids between extra- and intracellular membranes. Some family members are also believed to transport lipophilic molecules from outer cell membrane to certain intracellular receptors such as PPAR. The FABPs are intracellular carriers that “solubilize” the endocannabinoid anandamide (AEA), transporting AEA to the breakdown by FAAH, and compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) are also discovered to bind human FABPs (1, 3, 5, and 7) that function as intracellular carriers, as THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. Levels of fatty-acid-binding protein have been shown to decline with ageing in the mouse brain, possibly contributing to age-associated decline in synaptic activity.
Family members.
Members of this family include:


Vicianin

Vicianin is a cyanogenic disaccharide.
The enzyme vicianin beta-glucosidase uses ("R")-vicianin and H2O to produce mandelonitrile and vicianose. It is found in seeds of "Vicia angustifolia".


Thermosome

A thermosome is a group II chaperonin protein complex that functions in archaea. It is the homolog of eukaryotic CCT. This group II chaperonin is an ATP-dependent chaperonin that is responsible for folding or refolding of incipient or denatured proteins. A thermosome has two rings, each consisting of eight subunits, stacked together to form a cylindrical shape with a large cavity at the center. The thermosome is also defined by its heterooligomeric nature. The complex consists of that alternate location within its two rings.
Being a Group II chaperonin, the thermosome has a similar structure to group I chaperonins. The main difference, however, lies in the existence of a helical protrusion in the thermosome which composes of a built-in lid of the hydrophilic cavity. Not only is thermosome ATP-dependent, but the mechanism in which thermosome shifts from open to close conformation is also temperature-dependent. The open conformation of the ATP-thermosome exists mainly at low temperatures. Whereas, the closed conformation of the thermosome occurs when heating to physiological temperature.