APOA5

Apolipoprotein A-V is a protein that in humans is encoded by the "APOA5" gene on chromosome 11. It is significantly expressed in liver. The protein encoded by this gene is an apolipoprotein and an important determinant of plasma triglyceride levels, a major risk factor for coronary artery disease. It is a component of several lipoprotein fractions including VLDL, HDL, chylomicrons. It is believed that apoA-V affects lipoprotein metabolism by interacting with LDL-R gene family receptors. Considering its association with lipoprotein levels, APOA5 is implicated in metabolic syndrome. The "APOA5" gene also contains one of 27 SNPs associated with increased risk of coronary artery disease.
Discovery.
The gene for apolipoprotein A5 (APOA5, gene ID 116519, OMIM accession number – 606368) was originally found by comparative sequencing of human and mice DNA as a last member of the gene cluster of apolipoproteins APOA1/APOC3/APOA4/APOA5, located on human chromosome 11 at position 11q23. The creation of two mice models (APOA5 transgenic and APOA5 knock-out) confirmed the important role of this gene in plasma triglyceride determination. The transgenic mice had lower and the knock-out mice higher levels of plasma triglycerides, while plasma cholesterol levels remained unchanged in both animal models. A Dutch group simultaneously described an identical gene as apolipoprotein which it is associated with the early phase of liver regeneration, but failed to recognise its important role in the determination of plasma triglyceride levels.
Structure.
Gene.
The "APOA5" gene resides on chromosome 11 at the band 11q23 and contains 4 exons and 3 introns. This gene uses alternate polyadenylation sites and is located proximal to the apolipoprotein gene cluster on chromosome 11q23.
Protein.
This protein belongs to the apolipoprotein A1/A4/E family and contains 2 coiled coil domains. Overall, APOA5 is predicted to have approximately 60% a-helical content. The mature APOA5 protein spans a length of 366 amino acid residues, of which 23 amino acids code for the signal peptide. The molecular mass of the precursor was calculated to be 41 kDa, while the mature APOA5 protein was calculated to be 39 kDa.
Tissue distribution.
In humans, APOA5 is expressed almost exclusively in the liver tissue; some minor expressions have also been detected in the small intestine. Nothing is known about the existence of the potential alternative splicing variants of this gene. In comparison with other apolipoproteins, plasma concentration of APOA5 is very low (less than 1 μg/mL). This suggests that it has more catalytic than structural functions, since there is less than one APOA5 molecule per one lipoprotein particle. APOA5 is associated predominantly with TG-rich lipoproteins (chylomicrons and VLDL) and has also been detected on HDL particles.
Function.
APOA5 mainly functions to influence plasma triglyceride levels. The first suggested mechanism supposes that APOA5 functions as an activator of lipoprotein lipase (which is a key enzyme in triglyceride catabolism) and, through this process, enhances the metabolism of TG-rich particles. The second is the possible effect of APOA5 on the secretion of VLDL particles, since APOA5 reduces hepatic production by inhibiting VLDL-particle production and assembly by binding to cellular membranes and lipids. Finally, the third possibility relates to the acceleration of the hepatic uptake of lipoprotein remnants and it has been shown that APOA5 binds to different members of the low-density lipoprotein receptor family. In addition to its TG-lowering effect, APOA5 also plays a significant role in modulating HDL maturation and cholesterol metabolism. Increased APOA5 levels were associated with skewed cholesterol distribution from VLDL to large HDL particles. APOA5 mRNA is upregulated during liver regeneration and this suggests that APOA5 serves a function in hepatocyte proliferation. It’s also reported that APOA5 could enhance insulin secretion in beta-cells and the cell surface midkine could be involved in APOA5 endocytosis.
Gene variability.
Within the APOA5 gene, a couple of important SNPs with a widely confirmed effect on plasma TG levels as well as rare mutations have been described. In Caucasians, the common variants are inherited mostly in three haplotypes, which are characterised by two SNPs, namely rs662799 (T-1131>C; in almost complete LD with A-3>G, where the minor allele is associated with about 50% lower gene expression) and rs3135506 (Ser19>Trp; C56>G; alters the signal peptide and influences APOA5 secretion into plasma). There are also a further three common variants (A-3>G, IVS+476 G>A and T1259>C) which are not necessary for haplotype characterisation.
Population frequencies of common APOA5 alleles exhibit large interethnic differences. For example, there are about 15% of carriers of the rs66299(C) allele among Caucasians, but the frequency could reach even between 40% and 50% among Asians. In contrast, the Trp19 allele is very rare in the Asian population (less than 1% of carriers) but is common in Caucasians (about 15% of carriers). Vice versa, important SNP (rs2075291, G553T, Gly185>Cys) with a population frequency of about 5% has been detected among Asians, but it is extremely rare among Caucasians. Sporadic publications refer to some other common polymorphisms, e.g. Val153>Met (rs3135507, G457A) and also suggest significant sex-dependent associations with plasma lipids. Rare variants within the APOA5 gene have been described in a couple of different populations. Among the “common mutations/rare SNPs”, one of the most characterised on a population level is the Ala315>Val exchange. Originally detected in patients with extreme TG levels over 10 mmo/L, it was also found in about 0.7% of the general population (mostly in individuals with normal TG values), which suggests a low penetrance of this variant. More than twenty other rare variants (mutations) have been described within the human APOA5 gene. They cover a wide spectrum that includes preliminary stop codons, amino acid changes as well as insertions and deletions. These mutations are generally associated with hypertriglyceridaemia, but penetration is usually not 100%. Individual mutations have been found mostly in one pedigree only.
But not all the SNPs have a detrimental effect on TG levels. A recent report, showed that, in Sardinian population, the missense mutation Arg282Ser in APOA5 gene, correlates with a decrease in TG levels. The authors believe that this point mutation is a mayor modulatory of TG values in this population.
Clinical significance.
In humans, plasma triglycerides such as triacylglycerols have been long debated as an important risk factor for not only cardiovascular disease but also for other relevant morbidities, such as cancer, renal disease, suicide, and all-cause mortality. The APOA5 gene was found by comparative sequencing of ~200 kbp of human and mice DNA as the last member of the gene cluster of apolipoproteins located on human chromosome 11 at 11q23. Two mouse transgenic mouse models (APOA5 transgenic and APOA5 knockout) confirmed the important role of this gene in plasma triglyceride levels of plasma triglycerides. Obesity and metabolic syndrome are both closely related to plasma triglyceride levels and APOA5. Recent meta-analyses suggest that the effect on metabolic syndrome development is more profound for rs662799 in Asian population and for rs3135506 for Europeans. Moreover, meta-analysis that focused on rs662799 and the risk of type 2 diabetes mellitus has suggested a significant association in Asian populations, but not in European populations.
As a risk factor.
Even though plasma concentration of APOA5 is very low, some studies have focused on the analysis of the potential association of this biochemical parameter with cardiovascular disease (CVD). This relationship remains controversial, as higher plasma levels of APOA5 in individuals with CVD disease have been found in some, but not in all studies.
Plasma lipids and cardiovascular disease.
The major effect of the apolipoprotein A5 gene (and its variants) is on plasma triglyceride levels. Minor alleles (C1131 and Trp19) are primarily associated with the elevation of plasma triglyceride levels. The most extensive information available has been drawn from Caucasian populations, particularly in relation to the rs662799 SNP. Here, one minor allele is associated with an approximate 0,25 mmol/L increase of plasma TG levels. A similar effect is associated with the Trp19 allele, even though it has not been confirmed by a huge number of studies. Original studies have further described that the strongest effect of APOA5 polymorphisms on plasma TG levels is observed among Hispanics, with only minor effects detected among Africans. Among Asians, the effect on plasma TG levels is similar that found among Caucasians. Generally, studies have suggested significant interethnic differences and in some cases sex-dependent associations as well.
Sporadic publications have also mentioned a weak but nonetheless significant effect of APOA5 variants on plasma HDL-cholesterol and non-HDL cholesterol levels.
Myocardial infarction.
A large meta-analysis of 101 studies confirmed a risk associated with the presence of the minor APOA5 allele -1131C and coronary heart disease. The odds ratio was 1.18 for every C allele. There are far fewer studies on the second common APOA5 polymorphism, Ser19>Trp, even though available studies have detected that its effect on plasma triglycerides is similar to C-1131>T. Nevertheless, the minor Trp allele is also associated with increased risk of CVD, and it seems that especially homozygotes and carriers of more minor alleles (both -1131C and 19Trp) are at higher risk of CVD.
Clinical Marker.
A multi-locus genetic risk score study based on a combination of 27 loci, including the APOA5 gene, identified individuals at increased risk for both incident and recurrent coronary artery disease events, as well as an enhanced clinical benefit from statin therapy. The study was based on a community cohort study (the Malmo Diet and Cancer study) and four additional randomized controlled trials of primary prevention cohorts (JUPITER and ASCOT) and secondary prevention cohorts (CARE and PROVE IT-TIMI 22).
BMI, metabolic syndrome.
Obesity and metabolic syndrome are both closely related to plasma triglyceride levels. Therefore, the focus on an association between APOA5 and BMI or metabolic syndrome is understandable. Available studies show that minor APOA5 alleles could be associated with an enhanced risk of obesity or metabolic syndrome development. However, genome wide studies have failed to prove that APOA5 is a gene associated with BMI values and/or obesity, so the effect could be far from clinically significant or at least significantly context-dependent.
Nutri-, acti- and pharmacogenetic associations.
Several studies have focused on changes of anthropometrical (body weight, BMI, WHR,…) or biochemical parameters (mostly plasma lipid levels) as a result of the interactions between common APOA5 variants and dietary habits (polyunsaturated fatty acid intake, n-3 and n-6 fatty acid intake, total fat and total energy intake, alcohol intake), dietary (lowering the energy intake) and/or physical activity interventions or dyslipidaemic (using statins or fenofibrate) treatment. Due to the high heterogeneity of the examined populations, differences in protocol and/or interventions used, the studies are difficult to directly compare and draw definitive conclusions. However, with caution, it could be concluded that carriers of the minor C-1131, Trp19, or T553 alleles are in some cases less prone to the positive effects of environmental and/or pharmacological interventions. Some papers suggest the importance of the interactions between APOA5 and other genes, especially with common APOE (OMIM acc. No. 107741) three allelic (E2, E3, and E4) polymorphism, in the modulation of plasma lipids. In these cases, the interaction between minor alleles of both genes seems to be of importance. In the general population, APOE4 seems to have the potential to diminish the effect of minor APOA5 rs662799 and rs3135506 alleles, especially in females. Interaction between APOE and APOA5 Ser19˃Trp has been suggested to play some role in the development of type III hyperlipidaemia. Further studies, in which interaction with APOA5 has been described, have included, for example, variants within FTO, lipoprotein lipase, USF-1 and FEN-1. They have also focused not only on plasma lipids, but on BMI values or hypertension as well.
Other roles.
Some other possible roles of APOA5 variants have been discussed, but generally these reports comprise only one or two papers – and first original papers with positive findings are usually not confirmed in second publications. These papers focus on the possible effect of different APOA5 variants on maternal height, longer foetal birth length, putative associations with plasma levels of C-reactive protein, LDL particle size and haemostatic markers. Despite the very low plasma concentration, variants within apolipoprotein A5 are potent determinants of plasma triglyceride levels. Minor alleles of three SNPs (rs662799, rs3135506, rs3135507) are associated with the higher risk of cardiovascular disease.


SIN3A

Paired amphipathic helix protein Sin3a is a protein that in humans is encoded by the "SIN3A" gene.
Function.
The protein encoded by this gene is a transcriptional regulatory protein. It contains paired amphipathic helix (PAH) domains, which are important for protein-protein interactions and may mediate repression by the Mad-Max complex.
Interactions.
SIN3A has been shown to interact with:


Mga (protein)

Mga is a DNA-binding protein that activates the expression of several important virulence genes in "Streptococcus pyogenes" (group A "Streptococcus", GAS) in response to changing environmental conditions. The family also contains VirR like proteins which match only at the C-terminus.
Mga is a wide-reaching regulator, affecting gene expression in over 10% of the "S. pyrogenes" genome. The other large regulator of virulence in GAS is the CovR/S two-component system, which affects the expression of approximately 15% of the genome. The two systems are linked through another protein, RivR, and a small non-coding RNA RivX.


3-hydroxy-2-methylquinolin-4-one 2,4-dioxygenase

In enzymology, a 3-hydroxy-2-methylquinolin-4-one 2,4-dioxygenase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are 3-hydroxy-2-methyl-1H-quinolin-4-one and O2, whereas its two products are N-acetylanthranilate and CO.
This enzyme belongs to the family of oxidoreductases, specifically those acting on single donors with O2 as oxidant and incorporation of two atoms of oxygen into the substrate (oxygenases). The oxygen incorporated need not be derived from O2. The systematic name of this enzyme class is 3-hydroxy-2-methyl-1H-quinolin-4-one 2,4-dioxygenase (CO-forming). This enzyme is also called (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase.


Iduronic acid

-Iduronic acid (IUPAC abbr.: IdoA) is the major uronic acid component of the glycosaminoglycans (GAGs) dermatan sulfate, and heparin. It is also present in heparan sulfate, although here in a minor amount relative to its carbon-5 epimer glucuronic acid.
IdoA is a hexapyranose sugar. Most hexapyranoses are stable in one of two chair conformations 1C4 or 4C1. -iduronate is different and adopts more than one solution conformation, with an equilibrium existing between three low-energy conformers. These are the 1C4 and 4C1 chair forms and an additional 2S0 skew-boat conformation.
IdoA may be modified by the addition of an "O"-sulfate group at carbon position 2 to form 2-"O"-sulfo--iduronic acid (IdoA2S).
In 2000, LK Hallak described the importance of this sugar in respiratory syncytial virus (RSV) infection. Dermatan sulfate and heparan sulfate were the only GAGs containing IdoA, and they were the only ones that inhibited RSV infection in cell culture.
When internally positioned within an oligosaccharide, the 1C4 and 2S0 conformations (shown below for IdoA2S) predominate.
Proton NMR spectroscopy can be used to track changes in the balance of this equilibrium.


Holin superfamily IV

The Holin superfamily IV is a superfamily of integral membrane transport proteins. It is one of the seven different holin superfamilies in total.
The Holin superfamily IV includes the TC families:
Superfamily IV includes four TC families, which includes members from "Firmicutes", "Actinobacteria" and "Fusobacteria". Their average sizes are (in amino acyl residues (aas)):
TC# 1.E.10 - 138 ± 6 aas
TC# 1.E.16 - 149 ± 15 aas
TC# 1.E.19 - 141 ± 12 aas
TC# 1.E.40 - 173 ± 149 aas
Thus, there is very little size variation among family members, except for family 40, which has members larger than the other proteins in this superfamily. All members of families 10 and 19 and many members of family 16 appear to have 3 transmembrane segments (TMSs), but some members of this last family are predicted to have 4 TMSs. The extra TMS in these proteins is at their N-termini.


Aurora inhibitor

Aurora kinases regulate cell cycle transit from G2 through cytokinesis and, thus, are targets in cancer therapy. There are three mammalian aurora kinase genes, encoding aurora A, B and C. Intense investigation has focused on aurora A and B as they appear to play a role in oncogenesis with aurora A identified as a low penetrance tumor susceptibility gene in mice and humans. Aurora kinases could be potential targets for novel small-molecule enzyme inhibitors.
Drug development.
A new approach to inhibiting cancer growth that shows great promise for structure-based drug development is targeting enzymes central to cellular mitosis. Aurora kinases, so named because the scattered mitotic spindles generated by mutant forms resemble the Aurora Borealis, have gained a great deal of attention as possible anticancer drug targets. The Aurora enzymes are particularly significant because they are involved in a direct path to the nucleosome by phosphorylating histone H3. Furthermore, Aurora kinases are known to be oncogenic and overexpressed in various forms of cancerous growth, including leukemia, colon cancer, prostate cancer and breast cancer tumors.
So far three Aurora-kinase inhibitors have been described: ZM447439, hesperadin and VX-680. The last is in advanced stages (Phase II clinical trial) of a joint drug development by Vertex Pharmaceuticals's VX-680 (Sausville, 234, last posted on 12/18/06) and Merck & Co., although the Phase II clinical trial was suspended in November, 2007 due to QT prolongation observed in one patient in Phase I trial.
Aurora structure.
The structure and active site of Aurora-2-adenosine complex has been determined. The hinge (yellow), glycine-rich loop (blue), and activation loop (red) are key features of the protein kinase fold involved in binding adenosine. The protein backbone atoms of residues Glu-211, Ala-213 in the hinge region of Aurora-2, and the sidechain of residue Trp-277, located in the activation loop, bind adenosine through specific hydrogen bonds. There are no hydrogen bonds between the 2'-OH or 3'-OH groups of the ribose moiety and Aurora-2. Residues Lys-162 and Asp-274 are essential for Aurora-2 kinase activity but do not hydrogen bond to each other as seen in crystal structures of several other protein kinases.


(R,R)-butanediol dehydrogenase

In enzymology, a (R,R)-butanediol dehydrogenase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are (R,R)-butane-2,3-diol and NAD, whereas its 3 products are (R)-acetoin, NADH, 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 (R,R)-butane-2,3-diol:NAD oxidoreductase. Other names in common use include butyleneglycol dehydrogenase, D-butanediol dehydrogenase, D-(−)-butanediol dehydrogenase, butylene glycol dehydrogenase, diacetyl (acetoin) reductase, D-aminopropanol dehydrogenase, D-aminopropanol dehydrogenase, 1-amino-2-propanol dehydrogenase, 2,3-butanediol dehydrogenase, D-1-amino-2-propanol dehydrogenase, (R)-diacetyl reductase, (R)-2,3-butanediol dehydrogenase, D-1-amino-2-propanol:NAD oxidoreductase, 1-amino-2-propanol oxidoreductase, and aminopropanol oxidoreductase. This enzyme participates in butanoic acid metabolism.


Onchidal

Onchidal is a naturally occurring neurotoxin produced as a defensive secretion by the mollusc "Onchidella binneyi" and several other related species in "Onchidella", a genus of small, air-breathing sea slugs. It acts as an irreversible acetylcholinesterase inhibitor, the same mechanism of action as that of the deadly nerve agents, however onchidal is not an organophosphorus or carbamate compound and bears little resemblance to other compounds of this nature.


Indican

Indican is a colourless organic compound, soluble in water, naturally occurring in "Indigofera" plants. It is a precursor of indigo dye.
Chemical reactions.
Common and significant reactions involving indican are as follows:
Medical significance.
Biosynthesis.
A reaction, similar to the above-mentioned is seen in the normal population, who excrete small amounts of the chemical in their urine. Normal urine reacting to hydrogen peroxide does at times produce a bluish tinge.
Tryptophan is first converted to indole (excreted in faeces), then to indican by bacteria in the gut. Indican, being water-soluble, is then excreted through the urine. Following absorption from the gut, indole is converted to "3-hydroxy indole (indoxyl or indican)" in the liver, where it is again then conjugated with sulfuric acid or glucoronic acid through normal xenobiotic metabolism pathways. It is then transported to the kidneys for excretion.
The enzyme "indoxyl esterase" has been found in humans and is involved in another pathway of chemical reactions involving indoxyl.
Pathology.
In individuals affected by the blue diaper syndrome, the patients exhibit a defect in tryptophan metabolism. Tryptophan is first converted to indole, then to indican by bacteria in the gut. Indican is then excreted into the urine and from there into the diaper where, upon exposure to air, it is converted to indigo blue dye due to oxidation by atmospheric oxygen.
Indican interferes with many commercial procedures for measuring total bilirubin which can be a problem for renal failure patients where blood indican levels are raised. It can cause gastrointestinal symptoms in patients where protein absorption is reduced - like Hartnup's disease, allowing for greater bacterial decomposition of the tryptophan to indole and its conversion to indican.


Gelatinase

Gelatinases are enzymes capable of degrading gelatin.
Gelatinases are expressed in several bacteria including "Pseudomonas aeruginosa" and "Serratia marcescens".
In humans, the gelatinases are matrix metalloproteinases MMP2 and MMP9.


2-hydroxy-3-oxoadipate synthase

In enzymology, a 2-hydroxy-3-oxoadipate synthase () is an enzyme that catalyzes the following chemical reaction:
The two substrates of this enzyme are 2-oxoglutarate and glyoxylate, whereas its two products are 2-hydroxy-3-oxoadipate and CO2.
This enzyme belongs to the family of transferases, specifically those transferring aldehyde or ketonic groups (transaldolases and transketolases, respectively). Other names in common use include 2-hydroxy-3-oxoadipate glyoxylate-lyase (carboxylating), alpha-ketoglutaric-glyoxylic carboligase, and oxoglutarate: glyoxylate carboligase. This enzyme participates in glyoxylate and dicarboxylate metabolism. It employs one cofactor, thiamin diphosphate.


Carveol dehydrogenase

In enzymology, a carveol dehydrogenase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are (-)-trans-carveol and NADP+, whereas its 3 products are (-)-carvone, 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 (-)-trans-carveol:NADP+ oxidoreductase. This enzyme is also called (-)-trans-carveol dehydrogenase. This enzyme participates in monoterpenoid biosynthesis and limonene and pinene degradation.


DD-carboxypeptidase

DD-carboxypeptidase may refer to:


ALOXE3

Epidermis-type lipoxygenase 3 (ALOXE3 or eLOX3) is a member of the lipoxygenase family of enzymes; in humans, it is encoded by the "ALOXE3" gene. This gene is located on chromosome 17 at position 13.1 where it forms a cluster with two other lipoxygenases, ALOX12B and ALOX15B. Among the human lipoxygenases, ALOXE3 is most closely (54% identity) related in amino acid sequence to ALOX12B. ALOXE3, ALOX12B, and ALOX15B are often classified as epidermal lipoxygenases, in distinction to the other three human lipoxygenases (ALOX5, ALOX12, and ALOX15), because they were initially defined as being highly or even exclusively expressed and functioning in skin. The epidermis-type lipoxygenases are now regarded as a distinct subclass within the multigene family of mammalian lipoxygenases with mouse Aloxe3 (also termed e-Lox-3) being the ortholog to human ALOXE3, mouse Alox12b being the ortholog to human ALOX12B (MIM 603741), and mouse Alox8 being the ortholog to human ALOX15B (MIM 603697)[supplied by OMIM]. ALOX12B and ALOXE3 in humans, Alox12b and Aloxe3 in mice, and comparable orthologs in other in other species are proposed to act sequentially in a multistep metabolic pathway that forms products that are structurally critical for creating and maintaining the skin's water barrier function.
Tissue distribution.
Immunologically detected ALOXE3 and ALOX12B in humans and Aloxe3 and Alox12b in mice have a similar tissue distribution in being highly expressed in the outer, differentiated layers of the epidermis; they co-localize at the surface of keratinocytes in the stratum granulosum of mouse skin and during mouse embryogenesis appear concurrently at the onset of skin development at day 15.5. ALOXE3 mRNA in humans was also detected at low levels in the pancreas, ovary, brain, testis, placenta, and some secretory epithelia. Aloxe3 and Alox12b mRNA was detected in the tongue, forestomach, trachea, brain, testis, and adipose tissue of mice and in the spinal cord of rats.
Activity.
Epidermal tissue.
ALOX12B, like most of the other lipoxygenases, possesses dioxygenase (EC 1.13.11) activity: it catalyzes the incorporate dioxygen (i.e. molecular oxygen [O2]) into a single substrate. Owing to this activity, the enzyme adds (O2) in the form of a hydroperoxyl (HO2) residue to arachidonic acid at its 12th carbon thereby forming 12("R")-hydroperoxy-5"Z",8"Z",10"E",14"Z"-icosatetraenoic acid (also termed 12("R")-HpETE or 12"R"-HpETE).
arachidonic acid + O2 formula_1 12"R"-HpETE
Hydroperoxy-containing polyunsaturated fatty acids (PUFAs) such as 12"R"-HETE readily breakdown through non-enzymatic transformations in which the two oxygen atoms of the hydroperoxy residue rearrange to form PUFAs containing one hydroxyl (also termed alcohol) residue and one epoxide residue. This transformation may occur in tissues or during tissue preparations with 12-HpETE to form Hepoxilins, i.e. epoxyalcohols of 12-HpETE that are of the A type (i.e. hepoxilin As, which contain an epoxy and alcohol residue separated from each other by a double (i.e. alkene) bond or, alternatively, B type (i.e. hepoxilin Bs, which contain epoxy and alcohol residues on adjacent carbons); these non-enxymatically formed products are a mixture of hydroxy and epoxy "R","S" stereoisomers and diastereomers. In addition to arachidonic acid, ALOX12B metabolizes linoleic acid (LA) to 9("R")-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9"R"-HpODE):
LA + O2 formula_19"R"-HpODE.
ALOXE3 is an atypical lipoxygenase in that under most but not all experimental conditions, it lacks the dioxygenase activity that converts PUFA to hydroperoxide metabolites; rather, it possess hepoxilin synthase (i.e. hydroperoxy isomerase) activity; that is, it converts hydroperoxy-containing PUFAs to hepoxilin-like epoxyalcohol products; these products, unlike those formed by non-enzymatic transformations, are specific isomers with just one form of the chiral hydroxy and epoxy residues. ALOX3E metabolizes 12"R"-HpETE to 8"R"-hydroxy-11"R",12"R"-epoxy-eicosatrienoic acid and metabolizes 9"R"-HpODE to products that contain either an epoxyalcohol or a ketone residue. It exhibits relatively weak activity in conducting this conversion on free 9"R"-HODE but stronger activity when 9"R"-HpODE is presented as its methyl ester. ALOXE3's primary function in epidermal tissue appears to be to metabolize the 9"R"-HpODE moiety that is not free but rather esterified to certain ceramide lipids.
LA is the most abundant fatty acid in the skin epidermis, being present mainly esterified to the omega-hydroxyl residue of amide-linked omega-hydroxylated very long chain fatty acids (VLCFAs) in a unique class of ceramides termed esterified omega-hydroxyacyl-sphingosine (EOS). EOS is an intermediate component in a proposed multi-step metabolic pathway which delivers VLCFAs to the cornified lipid envelop in the skin's Stratum corneum; the presence of these wax-like, hydrophobic VLCFAs is needed to maintain the skin's integrity and functionality as a water barrier (see Lung microbiome#Role of the epithelial barrier). ALOX12B metabolizes the LA in EOS to its 9"R"-hydroperoxy derivative which ALOXE3 then converts to three ceramide-esterified products: a) 9"R",10"R"-trans-epoxide,13"R"-hydroxy-10"E"-octadecenoic acid, b) 9-keto-10"E",12"Z"-octadecadienoic acid, and c) 9"R",10"R"-trans-epoxy-13-keto-11"E"-octadecenoic acid. The ALOX12B/ALOE3-oxidized products, it is proposed, signal for their hydrolysis (i.e. removal) from EOS; this allows the multi-step metabolic pathway to proceed in delivering the VLCFAs to the cornified lipid envelop in the skin's Stratum corneum.
Other tissues.
AloxE3 appears responsible for forming hepoxilins A and/or B from 12"R"-HpETE in the spinal fluids of rats and ALOXE3 is proposed to be responsible for the formation of these hepoxilins in various human tissues although the presence and activity of ALOXE3 in many of these hepoxilin-forming tissues has not yet been demonstrated.
Spinal Aloxe3, apparently through its ability to make hepoxilins, appears responsible for the hyperalgesia which accompanies inflammation in rats.
Aloxe3 appears necessary and sufficient for the differentiation of mouse 3T3-L1 fibroblast cells into adipocytes (i.e. fat cells); the function of Aloxe3 in this differentiation appears to be to its metabolism 12"R"-HpETE into hepoxilins A3 or B3 which directly activate(s) Peroxisome proliferator-activated receptor gamma which in turn initiates the expression of adipocyte-differentiation genes.
Clinical significance.
Congenital ichthyosiform erythrodema.
Deletions of "Alox12b" or "Aloxe3" genes by gene knockout in mice cause a congenital scaly skin disease which is characterized by a greatly reduced skin water barrier function and other features found in the autosomal recessive nonbullous Congenital ichthyosiform erythroderma (ARCI) disease of humans.; homozygous recessive deleterious mutations in ALOXE3 or ALOX12B are likewise causes, albeit rare, of this congenital disease in humans. ARCI refers to nonsyndromic (i.e. not associated with other signs or symptoms) congenital Ichthyosis including Harlequin-type ichthyosis, Lamellar ichthyosis, and Congenital ichthyosiform erythroderma. ARCI has an incidence of about 1/200,000 in European and North American populations; 40 different mutations in "ALOX12B" and 13 different mutations in "ALOXE3" genes account for a total of about 10% of ARCI cases; these mutations are homozygous recessive (see Dominance (genetics)), cause a total loss of ALOX12B or ALOXE3 function (see mutations), and can be associated with any of the three cited forms of the disease.
Hepoxilin synthase.
In mice lacking Aloxe3 activity due to gene knockout of the "Alox3" gene, levels in skin of hepoxilins A3 and B3, as well as their metabolites, trioxilins A3 and B3, are greatly reduced. Furthermore, rat Aloxe3 has been implicated in the production of hepoxilin B3 in studies that transfected its gene into cultured HEK 293 cells and similarly implicated in the inflammation-induced production of hepoxilin B3 in the spine of rats as well as the perception of pain (i.e. allodynia) by these animals using pharmacological inhibitor and siRNA-based gene knockdown studies. Finally, cultured human skin cells, which are rich in ALOXE3 readily convert arachidonic acid as well as 12"S"-hydroperoxy-eicosatetraenoic acid to Hepoxilin B3; this production, in keeping with the higher content of ALOXE3, is far greater in the skin cells isolated from subjects with psoriasis. These results suggest that ALOXE3 and its orthologs contribute greatly to or are the hepoxylin synthase activity responsible for producing bioactive hepoxilins (see hepoxilin) in the skin and other ALOXE3/ortholog-rich tissues of mammals, possibly including humans.
Other possible clinical significances.
The distribution of ALOXE3 and Aloxe3 (see Tissue distribution, above) suggests that these lipoxygenases may serve functions not only in the skin but also in other tissues. The studies reported in the above "Activities, Other tissues", subsection allow that the pain perception and adipocyte differentiation activities of Aloxe3 in rodents might also occur in humans.
Toxicity.
Interuterine delivery of e-Lox-3 to mice at gestational day 14.5 resulted in fetal growth restriction and intrauterine death apparently due to a strongly negative effect on placental development.


4-trimethylammoniobutyraldehyde dehydrogenase

In enzymology, a 4-trimethylammoniobutyraldehyde dehydrogenase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are 4-trimethylammoniobutanal, NAD+, and H2O, whereas its 3 products are 4-trimethylammoniobutanoate, NADH, and H+.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the aldehyde or oxo group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is 4-trimethylammoniobutanal:NAD+ 1-oxidoreductase. Other names in common use include 4-trimethylaminobutyraldehyde dehydrogenase, and 4-N-trimethylaminobutyraldehyde dehydrogenase. This enzyme participates in lysine degradation and carnitine biosynthesis.


AS3MT

Arsenite methyltransferase is an enzyme that in humans is encoded by the "AS3MT" gene.


Glycoside hydrolase family 35

In molecular biology, glycoside hydrolase family 35 is a family of glycoside hydrolases.
Glycoside hydrolases are a widespread group of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycoside hydrolases, based on sequence similarity, has led to the definition of >100 different families. This classification is available on the CAZy web site, and also discussed at CAZypedia, an online encyclopedia of carbohydrate active enzymes.
Glycoside hydrolase family 35 CAZY GH_35 comprises enzymes with only one known activity; beta-galactosidase (). Mammalian beta-galactosidase is a lysosomal enzyme (gene GLB1) which cleaves the terminal galactose from gangliosides, glycoproteins, and glycosaminoglycans and whose deficiency is the cause of the genetic disease Gm(1) gangliosidosis (Morquio disease type B).


Alpha-glucosidase inhibitor

Alpha-glucosidase inhibitors (AGIs) are oral anti-diabetic drugs used for diabetes mellitus type 2 that work by preventing the digestion of carbohydrates (such as starch and table sugar). Carbohydrates are normally converted into simple sugars (monosaccharides) by alpha-glucosidase enzymes present on cells lining the intestine, enabling monosaccharides to be absorbed through the intestine. Hence, alpha-glucosidase inhibitors reduce the impact of dietary carbohydrates on blood sugar.
Examples and differences.
Examples of alpha-glucosidase inhibitors include:
Even though the drugs have a similar mechanism of action, there are subtle differences between acarbose and miglitol. Acarbose is an oligosaccharide, whereas miglitol resembles a monosaccharide. Miglitol is fairly well absorbed by the body, as opposed to acarbose. Moreover, acarbose inhibits pancreatic alpha-amylase in addition to alpha-glucosidase.
Natural alpha glucosidase inhibitors.
There are a large number of natural products with alpha-glucosidase inhibitor action.
For example, research has shown the culinary mushroom Maitake ("Grifola frondosa") has a hypoglycemic effect. The reason Maitake lowers blood sugar is because the mushroom naturally contains an alpha glucosidase inhibitor.
Another plant attracting a lot of attention is "Salacia oblonga". /
Clinical use.
Alpha-glucosidase inhibitors are used to establish greater glycemic control over hyperglycemia in diabetes mellitus type 2, particularly with regard to postprandial hyperglycemia. They may be used as monotherapy in conjunction with an appropriate diabetic diet and exercise, or they may be used in conjunction with other anti-diabetic drugs.
In patients with diabetes mellitus type 1, Alpha-glucosidase inhibitors use has not been officially approved by the Food and Drug Administration but some data exists on the effectiveness in this population, showing potential benefits weighted against an increased risk of hypoglycemia.
Mechanism of action.
Alpha-glucosidase inhibitors are saccharides that act as competitive inhibitors of enzymes needed to digest carbohydrates: specifically alpha-glucosidase enzymes in the brush border of the small intestines. The membrane-bound intestinal alpha-glucosidases hydrolyze oligosaccharides, trisaccharides, and disaccharides to glucose and other monosaccharides in the small intestine.
Acarbose also blocks pancreatic alpha-amylase in addition to inhibiting membrane-bound alpha-glucosidases. Pancreatic alpha-amylase hydrolyzes complex starches to oligosaccharides in the lumen of the small intestine.
Inhibition of these enzyme systems reduces the rate of digestion of carbohydrates. Less glucose is absorbed because the carbohydrates are not broken down into glucose molecules. In diabetic patients, the short-term effect of these drugs therapies is to decrease current blood glucose levels: the long-term effect is a small reduction in hemoglobin A1c level.
Dosing.
Since alpha-glucosidase inhibitors are competitive inhibitors of digestive enzymes, they must be taken at the start of main meals to have maximal effect. Their effects on blood sugar levels following meals will depend on the amount of complex carbohydrates in the meal.
Side effects and precautions.
Since alpha-glucosidase inhibitors prevent the degradation of complex carbohydrates into glucose, the carbohydrates will remain in the intestine. In the colon, bacteria will digest the complex carbohydrates, thereby causing gastrointestinal side effects such as flatulence and diarrhea. Since these effects are dose-related, it is generally advised to start with a low dose and gradually increase the dose to the desired amount.
Pneumatosis cystoides intestinalis is another reported side effect.
If a patient using an alpha-glucosidase inhibitor suffers from an episode of hypoglycemia, the patient should eat something containing monosaccharides, such as glucose tablets. Since the drug will prevent the digestion of polysaccharides (or non-monosaccharides), non-monosaccharide foods may not effectively reverse a hypoglycemic episode in a patient taking an alpha-glucosidase inhibitor.


Peptide-aspartate beta-dioxygenase

In enzymology, a peptide-aspartate beta-dioxygenase (), a member of the alpha-ketoglutarate-dependent hydroxylases superfamily, is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are peptide-L-aspartate, 2-oxoglutarate, and O2, whereas its 3 products are peptide-3-hydroxy-L-aspartate, succinate, and CO2.
It employs one cofactor, iron.
Nomenclature.
This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with 2-oxoglutarate as one donor, and incorporation of one atom o oxygen into each donor. The systematic name of this enzyme class is peptide-L-aspartate,2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating). Other names in common use include aspartate beta-hydroxylase, and aspartylpeptide beta-dioxygenase.


SLIT3

Slit homolog 3 protein is a protein that in humans is encoded by the "SLIT3" gene.


Indane-5-sulfonamide

Indane-5-sulfonamide is the base structure of indanesulfonamides. Indane-5-sulfonamide is a carbonic anhydrase inhibitor.
Notes.
Carbonic anhydrase inhibitors: binding of indanesulfonamides to the human isoform II


Dissolving pulp

Dissolving pulp, also called dissolving cellulose, is bleached wood pulp or cotton linters that has a high cellulose content (> 90%). It has special properties including a high level of brightness and uniform molecular-weight distribution. This pulp is manufactured for uses that require a high chemical purity, and particularly low hemicellulose content, since the chemically similar hemicellulose can interfere with subsequent processes. Dissolving pulp is so named because it is not made into paper, but dissolved either in a solvent or by derivatization into a homogeneous solution, which makes it completely chemically accessible and removes any remaining fibrous structure. Once dissolved, it can be spun into textile fibers (viscose or Lyocell), or chemically reacted to produce derivatized celluloses, such cellulose triacetate, a plastic-like material formed into fibers or films, or cellulose ethers such as methyl cellulose, used as a thickener.
Manufacture.
Pulpwood.
Dissolving pulp is mainly produced chemically from pulpwood in a process that has a low yield (30 - 35% of the wood). This makes up of about 85 - 88% of the production. Dissolving pulp is made from the sulfite process or the kraft process with an acid prehydrolysis step to remove hemicelluloses. For the highest quality, it should be derived from fast-grown hardwoods with low non-cellulose content.
The sulfite process produces pulp with a cellulose content up to 92 percent. It can use ammonium, calcium, magnesium or sodium as a base. The prehydrolysis sulfate process produces pulp with a cellulose content up to 96%.
Special alkaline purification treatments can yield even higher cellulose levels: up to 96 percent for the sulfite process and up to 98 percent for the sulfate process.
Cotton linters.
A minor part is produced from the shortest cotton linters, normally second cut. These are washed mechanically and chemically to remove proteins, waxes, pectins and other polysaccharides. This is bleached to get the required brightness. Dissolving pulp from cellulose linters gives the purest cellulose and is used to manufacture acetate plastics and high-viscosity cellulose ethers.
Applications.
Dissolving pulp is used in production of regenerated cellulose. In the regenerated cellulose process the cellulose is converted to cellulose xanthate which dissolves easily in caustic soda. The resulting viscous liquid can be extruded through spinnerettes and regenerated as man-made fibres. Cellulose can also be dissolved in some organic solvents directly and processed to regenerate the cellulose fibres in different forms. The lyocell process uses an amine oxide to dissolve cellulose and Tencel is the only commercial example of this direct-dissolution process, which unlike the viscose process is pollution-free.
The 90-92% cellulose content sulfite pulps are used mostly to make textiles (like rayon) and cellophane. The 96-% cellulose content sulfate pulps are used to make rayon yarn for industrial products such as tire cord, rayon staple for high-quality fabrics, and various acetate and other specialty products.
As a raw material of cellulose derivatives, dissolving pulp is used in carboxymethyl cellulose (CMC), methyl cellulose (MC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), etc.
Since dissolving pulp is highly refined, it is a product of high whiteness with few impurities making it suitable in specialty paper-related products such as filter paper and vulcanized fibre.
Cellulose powder is dissolving pulp that has undergone acid hydrolysis, been mechanically disintegrated and made into fine powder.
This pulp is used as a filler for urea-formaldehyde resins and melamine resin products.


Pair-rule gene

A pair-rule gene is a type of gene involved in the development of the segmented embryos of insects. Pair-rule genes are expressed as a result of differing concentrations of gap gene proteins, which encode transcription factors controlling pair-rule gene expression. Pair-rule genes are defined by the effect of a mutation in that gene, which causes the loss of the normal developmental pattern in alternating segments.
Pair-rule genes were first described by Christiane Nüsslein-Volhard and Eric Wieschaus in 1980. They used a genetic screen to identify genes required for embryonic development in the fruit fly "Drosophila melanogaster". In normal unmutated "Drosophila," each segment produces bristles called denticles in a band arranged on the side of the segment closer to the head (the anterior). They found five genes – "even-skipped, hairy, odd-skipped, paired" and "runt" – where mutations caused the deletion of a particular region of every alternate segment. For example, in "even-skipped," the denticle bands of alternate segments are missing, which results in an embryo having half the number of denticle bands. Later work identified more pair-rule genes in the "Drosophila" early embryo – "fushi tarazu", "odd-paired" and "sloppy paired".
Once the pair-rule genes had been identified at the molecular level it was found that each gene is expressed in alternate "parasegments" – regions in the embryo that are closely related to segments, but are slightly out of register. Each parasegment includes the posterior part of one (future) segment, and an anterior part of the next (more posterior) segment. The bands of expression of the pair-rule genes correspond to the regions missing in the mutant. The expression of the pair-rule genes in bands is dependent both upon direct regulation by the gap genes and on regulatory interactions between the pair-rule genes themselves.


ADP-specific glucokinase

In enzymology, an ADP-specific glucokinase () also known as ADP-dependent glucokinase is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are ADP and D-glucose, whereas its two products are AMP and D-glucose 6-phosphate.
This enzyme belongs to the family of transferases, to be specific those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor.
In humans, the ADP-dependent glucokinase is encoded by the "ADPGK" gene.
Structural studies.
As of late 2007, only one structure has been solved for this class of enzymes, with the PDB accession code .


Restriction enzyme

A restriction enzyme, restriction endonuclease, or " restrictase " is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.
These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up "foreign" DNA in a process called "restriction digestion"; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.
Over 3,000 restriction enzymes have been studied in detail, and more than 800 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a vital tool in molecular cloning.
History.
The term restriction enzyme originated from the studies of phage λ, a virus that infects bacteria, and the phenomenon of host-controlled restriction and modification of such bacterial phage or bacteriophage. The phenomenon was first identified in work done in the laboratories of Salvador Luria, Weigle and Giuseppe Bertani in the early 1950s. It was found that, for a bacteriophage λ that can grow well in one strain of "Escherichia coli", for example "E. coli" C, when grown in another strain, for example "E. coli" K, its yields can drop significantly, by as much as 3-5 orders of magnitude. The host cell, in this example "E. coli" K, is known as the restricting host and appears to have the ability to reduce the biological activity of the phage λ. If a phage becomes established in one strain, the ability of that phage to grow also becomes restricted in other strains. In the 1960s, it was shown in work done in the laboratories of Werner Arber and Matthew Meselson that the restriction is caused by an enzymatic cleavage of the phage DNA, and the enzyme involved was therefore termed a restriction enzyme.
The restriction enzymes studied by Arber and Meselson were type I restriction enzymes, which cleave DNA randomly away from the recognition site. In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated and characterized the first type II restriction enzyme, "Hind"II, from the bacterium "Haemophilus influenzae". Restriction enzymes of this type are more useful for laboratory work as they cleave DNA at the site of their recognition sequence and are the most commonly used as a molecular biology tool. Later, Daniel Nathans and Kathleen Danna showed that cleavage of simian virus 40 (SV40) DNA by restriction enzymes yields specific fragments that can be separated using polyacrylamide gel electrophoresis, thus showing that restriction enzymes can also be used for mapping DNA. For their work in the discovery and characterization of restriction enzymes, the 1978 Nobel Prize for Physiology or Medicine was awarded to Werner Arber, Daniel Nathans, and Hamilton O. Smith. The discovery of restriction enzymes allows DNA to be manipulated, leading to the development of recombinant DNA technology that has many applications, for example, allowing the large scale production of proteins such as human insulin used by diabetic patients.
Origins.
Restriction enzymes likely evolved from a common ancestor and became widespread via horizontal gene transfer. In addition, there is mounting evidence that restriction endonucleases evolved as a selfish genetic element.
Recognition site.
Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences can also be classified by the number of bases in its recognition site, usually between 4 and 8 bases, and the number of bases in the sequence will determine how often the site will appear by chance in any given genome, e.g., a 4-base pair sequence would theoretically occur once every 4^4 or 256bp, 6 bases, 4^6 or 4,096bp, and 8 bases would be 4^8 or 65,536bp. Many of them are palindromic, meaning the base sequence reads the same backwards and forwards. In theory, there are two types of palindromic sequences that can be possible in DNA. The "mirror-like" palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backward on a single strand of DNA, as in GTAATG. The "inverted repeat" palindrome is also a sequence that reads the same forward and backward, but the forward and backward sequences are found in complementary DNA strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG). Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes.
EcoRI digestion produces "sticky" ends,
whereas SmaI restriction enzyme cleavage produces "blunt" ends:
Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (5' end or 3' end) of a sticky-end "overhang" of an enzyme restriction.
Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the same location are known as isoschizomers.
Types.
Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence. DNA sequence analyses of restriction enzymes however show great variations, indicating that there are more than four types. All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements, as summarised below:
Type l.
Type I restriction enzymes were the first to be identified and were first identified in two different strains (K-12 and B) of "E. coli". These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site. Cleavage at these random sites follows a process of DNA translocation, which shows that these enzymes are also molecular motors. The recognition site is asymmetrical and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a non-specific spacer of about 6–8 nucleotides. These enzymes are multifunctional and are capable of both restriction digestion and modification activities, depending upon the methylation status of the target DNA. The cofactors S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP), and magnesium (Mg2+) ions, are required for their full activity. Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction digestion; HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity), and HsdS is important for specificity of the recognition (DNA-binding) site in addition to both restriction digestion (DNA cleavage) and modification (DNA methyltransferase) activity.
Type II.
Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They form homodimers, with recognition sites that are usually undivided and palindromic and 4–8 nucleotides in length. They recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity—they usually require only Mg2+ as a cofactor. These enzymes cleave the phosphodiester bond of double helix DNA. It can either cleave at the center of both strands to yield a blunt end, or at a staggered position leaving overhangs called sticky ends. These are the most commonly available and used restriction enzymes. In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes. These subgroups are defined using a letter suffix.
Type IIB restriction enzymes (e.g., BcgI and BplI) are multimers, containing more than one subunit. They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg2+ cofactors. Type IIE restriction endonucleases (e.g., NaeI) cleave DNA following interaction with two copies of their recognition sequence. One recognition site acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or improves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both sequences at the same time. Type IIG restriction endonucleases (e.g., RM.Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active. Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA. Type IIS restriction endonucleases (e.g., "Fok"I) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites; this characteristic is widely used to perform in-vitro cloning techniques such as Golden Gate cloning. These enzymes may function as dimers. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits. Some recognize palindromic sequences while others have asymmetric recognition sites.
Type III.
Type III restriction enzymes (e.g., EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20–30 base pairs after the recognition site. These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction digestion, respectively. They are components of prokaryotic DNA restriction-modification mechanisms that protect the organism against invading foreign DNA. Type III enzymes are hetero-oligomeric, multifunctional proteins composed of two subunits, Res () and Mod (). The Mod subunit recognises the DNA sequence specific for the system and is a modification methyltransferase; as such, it is functionally equivalent to the M and S subunits of type I restriction endonuclease. Res is required for restriction digestion, although it has no enzymatic activity on its own. Type III enzymes recognise short 5–6 bp-long asymmetric DNA sequences and cleave 25–27 bp downstream to leave short, single-stranded 5' protrusions. They require the presence of two inversely oriented unmethylated recognition sites for restriction digestion to occur. These enzymes methylate only one strand of the DNA, at the N-6 position of adenosyl residues, so newly replicated DNA will have only one strand methylated, which is sufficient to protect against restriction digestion. Type III enzymes belong to the beta-subfamily of N6 adenine methyltransferases, containing the nine motifs that characterise this family, including motif I, the AdoMet binding pocket (FXGXG), and motif IV, the catalytic region (S/D/N (PP) Y/F).
Type IV.
Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of "E. coli".
Type V.
Type V restriction enzymes (e.g., the cas9-gRNA complex from CRISPRs) utilize guide RNAs to target specific non-palindromic sequences found on invading organisms. They can cut DNA of variable length, provided that a suitable guide RNA is provided. The flexibility and ease of use of these enzymes make them promising for future genetic engineering applications.
Artificial restriction enzymes.
Artificial restriction enzymes can be generated by fusing a natural or engineered DNA-binding domain to a nuclease domain (often the cleavage domain of the type IIS restriction enzyme "Fok"I). Such artificial restriction enzymes can target large DNA sites (up to 36 bp) and can be engineered to bind to desired DNA sequences. Zinc finger nucleases are the most commonly used artificial restriction enzymes and are generally used in genetic engineering applications, but can also be used for more standard gene cloning applications. Other artificial restriction enzymes are based on the DNA binding domain of TAL effectors.
In 2013, a new technology CRISPR-Cas9, based on a prokaryotic viral defense system, was engineered for editing the genome, and it was quickly adopted in laboratories. For more detail, read CRISPR (Clustered regularly interspaced short palindromic repeats).
In 2017 a group from University of Illinois reported using an Argonaute protein taken from "Pyrococcus furiosus" (PfAgo) along with guide DNA to edit DNA "in vitro" as artificial restriction enzymes.
Artificial ribonucleases that act as restriction enzymes for RNA have also been developed. A PNA-based system, called a PNAzyme, has a Cu(II)-2,9-dimethylphenanthroline group that mimics ribonucleases for specific RNA sequence and cleaves at a non-base-paired region (RNA bulge) of the targeted RNA formed when the enzyme binds the RNA. This enzyme shows selectivity by cleaving only at one site that either does not have a mismatch or is kinetically preferred out of two possible cleavage sites.
Nomenclature.
Since their discovery in the 1970s, many restriction enzymes have been identified; for example, more than 3500 different Type II restriction enzymes have been characterized. Each enzyme is named after the bacterium from which it was isolated, using a naming system based on bacterial genus, species and strain. For example, the name of the EcoRI restriction enzyme was derived as shown in the box.
Applications.
Isolated restriction enzymes are used to manipulate DNA for different scientific applications.
They are used to assist insertion of genes into plasmid vectors during gene cloning and protein production experiments. For optimal use, plasmids that are commonly used for gene cloning are modified to include a short "polylinker" sequence (called the multiple cloning site, or MCS) rich in restriction enzyme recognition sequences. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA, since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a DNA ligase.
Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single-nucleotide polymorphisms (SNPs). This is however only possible if a SNP alters the restriction site present in the allele. In this method, the restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by gel electrophoresis. In general, alleles with correct restriction sites will generate two visible bands of DNA on the gel, and those with altered restriction sites will not be cut and will generate only a single band. A DNA map by restriction digest can also be generated that can give the relative positions of the genes. The different lengths of DNA generated by restriction digest also produce a specific pattern of bands after gel electrophoresis, and can be used for DNA fingerprinting.
In a similar manner, restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot. This technique allows researchers to identify how many copies (or paralogues) of a gene are present in the genome of one individual, or how many gene mutations (polymorphisms) have occurred within a population. The latter example is called restriction fragment length polymorphism (RFLP).
Artificial restriction enzymes created by linking the "Fok"I DNA cleavage domain with an array of DNA binding proteins or zinc finger arrays, denoted zinc finger nucleases (ZFN), are a powerful tool for host genome editing due to their enhanced sequence specificity. ZFN work in pairs, their dimerization being mediated in-situ through the "Fok"I domain. Each zinc finger array (ZFA) is capable of recognizing 9–12 base pairs, making for 18–24 for the pair. A 5–7 bp spacer between the cleavage sites further enhances the specificity of ZFN, making them a safe and more precise tool that can be applied in humans. A recent Phase I clinical trial of ZFN for the targeted abolition of the CCR5 co-receptor for HIV-1 has been undertaken.
Others have proposed using the bacteria R-M system as a model for devising human anti-viral gene or genomic vaccines and therapies since the RM system serves an innate defense-role in bacteria by restricting tropism by bacteriophages. There is research on REases and ZFN that can cleave the DNA of various human viruses, including HSV-2, high-risk HPVs and HIV-1, with the ultimate goal of inducing target mutagenesis and aberrations of human-infecting viruses. The human genome already contains remnants of retroviral genomes that have been inactivated and harnessed for self-gain. Indeed, the mechanisms for silencing active L1 genomic retroelements by the three prime repair exonuclease 1 (TREX1) and excision repair cross complementing 1(ERCC) appear to mimic the action of RM-systems in bacteria, and the non-homologous end-joining (NHEJ) that follows the use of ZFN without a repair template.
Examples.
Examples of restriction enzymes include:
Key:
N = C or G or T or A
W = A or T


2,3-diaminopropionate N-oxalyltransferase

In enzymology, a 2,3-diaminopropionate N-oxalyltransferase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are oxalyl-CoA and L-2,3-diaminopropanoate, whereas its two products are CoA and N3-oxalyl-L-2,3-diaminopropanoate.
This enzyme belongs to the family of transferases, specifically those acyltransferases transferring groups other than aminoacyl groups. The systematic name of this enzyme class is oxalyl-CoA:L-2,3-diaminopropanoate N3-oxalyltransferase. Other names in common use include oxalyldiaminopropionate synthase, ODAP synthase, oxalyl-CoA:L-alpha,beta-diaminopropionic acid oxalyltransferase, oxalyldiaminopropionic synthase, and oxalyl-CoA:L-2,3-diaminopropanoate 3-N-oxalyltransferase.


Cholesterol oxidase

In enzymology, a cholesterol oxidase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are cholesterol and O2, whereas its two products are cholest-4-en-3-one and H2O2.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with oxygen as acceptor. The systematic name of this enzyme class is cholesterol:oxygen oxidoreductase. Other names in common use include cholesterol- O2 oxidoreductase, 3beta-hydroxy steroid oxidoreductase, and 3beta-hydroxysteroid:oxygen oxidoreductase. This enzyme participates in bile acid biosynthesis.
The substrate-binding domain found in some bacterial cholesterol oxidases is composed of an eight-stranded mixed beta-pleated sheet and six alpha-helices. This domain is positioned over the isoalloxazine ring system of the FAD cofactor bound by the FAD-binding domain and forms the roof of the active site cavity, allowing for catalysis of oxidation and isomerisation of cholesterol to cholest-4-en-3-one.
Structural studies.
As of late 2007, 14 structures have been solved for this class of enzymes, with PDB accession codes , , , , , , , , , , , , , and .


L-isoaspartyl methyltransferase

Protein L-isoaspartyl methyltransferase (PIMT, PCMT), also called S-adenosyl-L-methionine:protein-L-isoaspartate O-methyltransferase, is an enzyme which recognizes and catalyzes the repair of damaged L-isoaspartyl and D-aspartyl groups in proteins. It is a highly conserved enzyme which is present in nearly all eukaryotes, archaebacteria, and Gram-negative eubacteria.
Function.
PIMT acts to transfer methyl groups from S-adenosyl-L-methionine to the alpha side chain carboxyl groups of damaged L-isoaspartyl and D-aspartyl amino acids. The enzyme takes the end methyl residue from the methionine side chain and adds it to the side chain carboxyl group of L-isoaspartate or D-aspartate to create a methyl ester. Subsequent nonenzymatic reactions result in a rapid transformation to L-succinimide, which is a precursor to aspartate and isoaspartate. The L-succinimide can then undergo nonenzymatic hydrolysis, which generates some repaired L-aspartyl residues as well as some L-isoaspartyl residues, which can then enter the cycle again for eventual conversion to the normal peptide linkage.
PIMT tends to act on proteins that have been non-enzymatically damaged due to age. By performing this repair mechanism, the enzyme helps to maintain overall protein integrity. This mechanism has been observed by several groups, and has been confirmed through experimental testing. In one report, PIMT was inhibited by adenosine dialdehyde. The results supported the proposed function of the enzyme, as the amount of abnormal L-aspartate residues increased when cells were treated with the indirect inhibitor, adenosine dialdehyde. Additionally, S-adenosylhomocysteine is known to be a competitive inhibitor of PIMT. When PIMT is not present in cells, the abnormal aspartyl residues accumulate, creating abnormal proteins that have been known to cause fatal progressive epilepsy in mice. It has been suggested that calmodulin may play a role in stimulating the function of PIMT, although the relationship between these two molecules has not been thoroughly explored. In addition to calmodulin, guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS) has been found to stimulate PIMT activity.
Structure.
The enzyme is present in human cytosol in two forms due to alternative splicing and differs among individuals in the population due to a single polymorphism at protein 119, either valine or isoleucine. The enzyme structure is described as a “doubly wound alpha/beta/alpha sandwich structure” which is quite consistent in all species analyzed thus far. If there is any difference in the sequences between different organisms it occurs in the regions connecting the three motifs in the sandwich structure, but the sequence of the individual motifs tends to be highly conserved. Researchers have found the active site to be in the loop between the beta structure and the second alpha helix and have determined it to be highly specific for isoaspartyl residues. For example, the residues found at the C-terminus of drosophila PIMT (dPIMT) are rotated 90 degrees so as to allow more space for a substrate to interact with the enzyme. In fact, dPIMT appears to alternate between this unique open conformation and the less open conformation common of PIMT in other organisms. Although possibly unrelated to this, increased levels of dPIMT in drosophila have been correlated with increase life expectancy in these organisms due to their importance in protein repair.


Heath bar

The Heath bar is a candy bar made of toffee, almonds, and milk chocolate. It was originally marketed by L. S. Heath beginning in 1914 and subsequently by Leaf Inc. and, since 1996, by Hershey.
Shaped as a thin, hard slab with a milk chocolate coating, the toffee originally contained sugar, butter, and almonds. Originally it was a small squarish bar weighing 1 ounce. It is similar to Hershey's Skor bar and Mondelēz's Daim bar.
In a 1987 popularity survey, the Heath bar ranked 56th nationally in the U.S. and 110th on the U.S. East Coast. It has become a popular add-in ingredient to ice cream, cookies, and other confections.
History.
In 1913, L. S. Heath, a school teacher, bought an existing confectionery shop in Robinson, Illinois as a likely business opportunity for his oldest sons, Bayard Heath and Everett Heath. There, in 1914, the brothers opened a combination candy store, ice cream parlor, and manufacturing operation.
With the success of the business, the elder Heath became interested in ice cream and opened a small dairy factory in 1915. His sons worked on expanding their confectionery business. At some point, they reportedly acquired a toffee recipe, via a traveling salesman, from a Greek confectioner in another part of the state. In 1928, they began marketing the toffee confection locally as "Heath English Toffee", proclaiming it "America's Finest".
In 1931, Bayard and Everett were persuaded by their father to sell the confectionery and work at his dairy. They brought their candy-making equipment with them and established a retail business there. The Heaths came up with the marketing idea of including their toffee confection on the dairy products order form taken around by the Heath dairy trucks: customers could then order Heath bars to be delivered along with milk and cottage cheese.
Early ads promoted Heath as a virtual health bar – only the best milk chocolate and almonds, creamery butter, and "pure sugar cane". The motto at the bottom of one ad read "Heath for better health!" The motto was surrounded by illustrations of milk, cream, butter, cheese, and ice cream and in a corner – a Heath bar and a bottle of soda. The soda may have been Pepsi, as the Heath Co. bottled the drink for a number of years.
The Heath bar grew in national popularity during the Depression, despite its 1-ounce size and the 5-cent price, equal to larger bars. Made by hand until 1942, the candy was manufactured consistently on a major commercial scale after the U.S. Army placed its first order of $175,000. The Heath bar had been found to have a very long shelf life: subsequently, the Army included it in soldiers' rations throughout World War II.
Popularity of the Heath bar grew after the war. However, the manufacturing process remained largely a hands-on, family-run operation: all four of L. S. Heath's sons, his two daughters, and several grandchildren were involved in the business. In the 1950s, the Heath Toffee Ice Cream Bar was developed, and eventually was franchised to other dairies.
In the 1960s, the huge national success of the Heath bar led to disagreements within the family, with at least one grandchild, Richard J. Heath, expelled from the business in 1969. He eventually published a book in 1995 entitled "Bittersweet: The Story of the Heath Candy Co."
In the 1970s, the company bought the registered trademark toffee ice cream flavoring formula called Butter Brickle from The Fenn Bros. Ice Cream and Candy Co. of Sioux Falls, South Dakota.
In 1989, with the diminishing and splintering of the Heath family, the business was sold to a Finnish company, Leaf, Inc., which in turn was acquired by Hershey in 1996. Hershey had initially created the Skor bar to compete with the Heath bar, before it bought out Leaf, Inc.
Since acquiring the product, Hershey has elongated the bar to align with its competition. It now weighs 1.4 ounces. Current ingredients are milk chocolate, sugar, vegetable oil, dairy butter (milk), almonds, salt, and soy lecithin. The wrapper's vintage brown color scheme has a small seal proclaiming Heath the "Finest Quality English Toffee".
Heath bars in other products.
Following the 1973 use of the candy bar as an ice-cream "mix-in" by Steve's Ice Cream, Heath bars became a significant ingredient in ice cream and other confections.
According to Ray Broekel in his 1982 book "The Great American Candy Bar Book", variations of the bar have included: Heath Milk Chocolate with Peanuts; Heath Milk Chocolate Toffee Crunch; Heath Milk Chocolate with Natural Cereal and Raisins; and the Double Heath bar. In the 1980s, a Heath Toffee Ice Cream Sandwich appeared, along with Heath Soft 'n Crunchy—a soft-serve ice cream.
Currently, other varieties of Heath bar-based confections include: Archway Cookies' "Heath Cookie"; "Heath Bar" Klondike bars; Baskin-Robbins' "Heath Bar Shake"; Dairy Queen's "Heath Bar Blizzard"; and Heath Bar flavored varieties of ice cream with a coffee or vanilla ice cream base. Ben and Jerry's produced a "Heath Bar Crunch" ice cream, which was renamed Vanilla Toffee Bar Crunch in 2014 when the company stopped using actual branded Heath bars.
Although the candy bar's original manufacturer, L. S. Heath, and subsequently Hershey have supported the incorporation of the candy bar into other confections by marketing a pre-shredded variety, many vendors hand-crumble the candy bars, finding the pre-crumbled variety to be "too small and too dusty".


LRRC48

Leucine-rich repeat-containing protein 48 is a protein that in humans is encoded by the "LRRC48" gene.


Dinosterol

Dinosterol is a type of steroid produced by several genera of dinoflagellates. It is a 4α-methyl sterol (4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol), a derivative of dinosterane, rarely found in other classes of protists.
This sterol and others have been considered as class-specific, being biomarkers for dinoflagellates, although dinosterol is produced in minor amounts by a small number of other phytoplankton, such as the marine diatom "Navicula speciosa". and Prymnesiophytes of the genus "Pavlova".
Dinosterols show similar abundances as dinocysts. Other studies found a nonlinear or did not find a direct link between dinocyst abundances and sterol concentrations.
Dinosterol has been used as indicator for dinoflagellate production in the Cariaco Basin.
Hydrogen isotope ratios in dinosterols can serve to reconstruct salinity semi-quantitatively.
Some studies have shown that certain dinoflagellates produce sterols that have the potential to serve as genera-specific biomarkers. Recent work showed that dinoflagellate genera, which formed discrete clusters in the 18S rDNA-based phylogeny, shared similar sterol compositions. This suggested that the sterol compositions of dinoflagellates are explained by the evolutionary history of this lineage.


Poly((R)-hydroxyalkanoic acid) depolymerase

Poly((R)-hydroxyalkanoic acid) depolymerase may refer to:


4-hydroxymuconic-semialdehyde dehydrogenase

In enzymology, a 4-hydroxymuconic-semialdehyde dehydrogenase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are 4-hydroxymuconic semialdehyde, NAD+, and H2O, whereas its 3 products are maleylacetate, NADH, and H+.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the aldehyde or oxo group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is 4-hydroxymuconic-semialdehyde:NAD+ oxidoreductase. This enzyme participates in gamma-hexachlorocyclohexane degradation.


Furylfuramide isomerase

In enzymology, a furylfuramide isomerase () is an enzyme that catalyzes the chemical reaction
Hence, this enzyme has one substrate, (E)-2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, and one product, (Z)-2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide.
This enzyme belongs to the family of isomerases, specifically cis-trans isomerases. The systematic name of this enzyme class is 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide cis-trans-isomerase. It has 2 cofactors: NAD+, and NADH.


FAD-AMP lyase (cyclizing)

In enzymology, a FAD-AMP lyase (cyclizing) () is an enzyme that catalyzes the chemical reaction
Hence, this enzyme has one substrate, FAD, and two products, AMP and riboflavin cyclic-4',5'-phosphate.
This enzyme belongs to the family of lyases, specifically the class of phosphorus-oxygen lyases. The systematic name of this enzyme class is FAD AMP-lyase (riboflavin-cyclic-4',5'-phosphate-forming). Other names in common use include FMN cyclase, and FAD AMP-lyase (cyclic-FMN-forming).


Mothers against decapentaplegic homolog 4

SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 (Deleted in Pancreatic Cancer-4) is a highly conserved protein present in all metazoans. It belongs to the SMAD family of transcription factor proteins, which act as mediators of TGF-β signal transduction. The TGFβ family of cytokines regulates critical processes during the lifecycle of metazoans, with important roles during embryo development, tissue homeostasis, regeneration, and immune regulation.
SMAD 4 belongs to the co-SMAD group ("common mediator" SMAD), the second class of the SMAD family. SMAD4 is the only known co-SMAD in most metazoans. It also belongs to the Darwin family of proteins that modulate members of the TGFβ protein superfamily, a family of proteins that all play a role in the regulation of cellular responses. Mammalian SMAD4 is a homolog of the "Drosophila" protein "Mothers against decapentaplegic" named Medea.
SMAD4 interacts with R-Smads, such as SMAD2, SMAD3, SMAD1, SMAD5 and SMAD8 (also called SMAD9) to form heterotrimeric complexes. Once in the nucleus, the complex of SMAD4 and two R-SMADS binds to DNA and regulates the expression of different genes depending on the cellular context. Intracellular reactions involving SMAD4 are triggered by the binding, on the surface of the cells, of growth factors from the TGFβ family. The sequence of intracellular reactions involving SMADS is called the SMAD pathway or the transforming growth factor beta (TGF-β) pathway since the sequence starts with the recognition of TGF-β by cells.
Gene.
In mammals, SMAD4 is coded by a gene located on chromosome 18. In humans, the "SMAD4" gene contains 54 829 base pairs and is located from pair n° 51,030,212 to pair 51,085,041 in the region 21.1 of the chromosome 18.
Protein.
SMAD4 is a 552 amino-acid polypeptide with a molecular weight of 60.439 Da. SMAD4 has two functional domains known as MH1 and MH2.
The complex of two SMAD3 (or of two SMAD2) and one SMAD4 binds directly to DNA though interactions of their MH1 domains. These complexes are recruited to sites throughout the genome by cell lineage-defining transcription factors (LDTFs) that determine the context-dependent nature of TGF-β action. Early insights into the DNA binding specificity of Smad proteins came from oligonucleotide binding screens, which identified the palindromic duplex 5'–GTCTAGAC–3' as a high affinity binding sequence for SMAD3 and SMAD4 MH1 domains. Other motifs have also been identified in promoters and enhancers. These additional sites contain the CAGCC motif and the GGC(GC)|(CG) consensus sequences, the latter also known as 5GC sites. The 5GC-motifs are highly represented as clusters of sites, in SMAD-bound regions genome-wide. These clusters can also contain CAG(AC)|(CC) sites. SMAD3/SMAD4 complex also binds to the TPA-responsive gene promoter elements, which have the sequence motif TGAGTCAG.
Structures.
MH1 domain complexes with DNA motifs.
The first structure of SMAD4 bound to DNA was the complex with the palindromic GTCTAGAC motif. Recently, the structures of SMAD4 MH1 domain bound to several 5GC motifs have also been determined. In all complexes, the interaction with the DNA involves a conserved β-hairpin present in the MH1 domain. The hairpin is partially flexible in solution and its high degree of conformational flexibility allows recognition of the different 5-bp sequences. Efficient interactions with GC-sites occur only if a G nucleotide is located deep in the major grove, and establishes hydrogen bonds with the guanidinium group of Arg81. This interaction facilitates a complementary surface contact between the Smad DNA-binding hairpin and the major groove of the DNA. Other direct interactions involve Lys88 and Gln83. The X-ray crystal structure of the "Trichoplax adhaerens" SMAD4 MH1 domains bound to the GGCGC motif indicates a high conservation of this interaction in metazoans.
MH2 domain complexes.
The MH2 domain, corresponding to the C-terminus, is responsible for receptor recognition and association with other SMADs. It interacts with the R-SMADS MH2 domain and forms heterodimers and heterotrimers. Some tumor mutations detected in SMAD4 enhance interactions between the MH1 and MH2 domains.
Nomenclature and origin of name.
SMADs are highly conserved across species, especially in the N terminal MH1 domain and the C terminal MH2 domain.
The SMAD proteins are homologs of both the "Drosophila" protein MAD and the "C. elegans" protein SMA. The name is a combination of the two. During "Drosophila" research, it was found that a mutation in the gene "MAD" in the mother repressed the gene "decapentaplegic" in the embryo. The phrase "Mothers against" was added, since mothers often form organizations opposing various issues, e.g. Mothers Against Drunk Driving (MADD), reflecting "the maternal-effect enhancement of dpp"; and based on a tradition of unusual naming within the research community. SMAD4 is also known as DPC4, JIP or MADH4.
Function and action mechanism.
SMAD4 is a protein defined as an essential effector in the SMAD pathway. SMAD4 serves as a mediator between extracellular growth factors from the TGFβ family and genes inside the cell nucleus. The abbreviation "co" in co-SMAD stands for "common mediator". SMAD4 is also defined as a signal transducer.
In the TGF-β pathway, TGF-β dimers are recognized by a transmembrane receptor, known as type II receptor. Once the type II receptor is activated by the binding of TGF-β, it phosphorylates a type I receptor. Type I receptor is also a cell surface receptor. This receptor then phosphorylates intracellular receptor regulated SMADS (R-SMADS) such as SMAD2 or SMAD3. The phosphorylated R-SMADS then bind to SMAD4. The R-SMADs-SMAD4 association is a heteromeric complex. This complex is going to move from the cytoplasm to the nucleus: it is the translocation. SMAD4 may form heterotrimeric, heterohexameric or heterodimeric complexes with R-SMADS.
SMAD4 is a substrate of the Erk/MAPK kinase and GSK3. The FGF (Fibroblast Growth Factor) pathway stimulation leads to Smad4 phosphorylation by Erk of the canonical MAPK site located at Threonine 277. This phosphorylation event has a dual effect on Smad4 activity. First, it allows Smad4 to reach its peak of transcriptional activity by activating a growth factor-regulated transcription activation domain located in the Smad4 linker region, SAD (Smad-Activation Domain). Second, MAPK primes Smad4 for GSK3-mediated phosphorylations that cause transcriptional inhibition and also generate a phosphodegron used as a docking site by the ubiquitin E3 ligase Beta-transducin Repeat Containing (beta-TrCP) that polyubiquitinates Smad4 and targets it for degradation in the proteasome. Smad4 GSK3 phosphorylations have been proposed to regulate the protein stability during pancreatic and colon cancer progression.
In the nucleus the heteromeric complex binds promoters and interact with transcriptional activators. SMAD3/SMAD4 complexes can directly bind the SBE. These associations are weak and require additional transcription factors such as members of the AP-1 family, TFE3 and FoxG1 to regulate gene expression.
Many TGFβ ligands use this pathway and subsequently SMAD4 is involved in many cell functions such as differentiation, apoptosis, gastrulation, embryonic development and the cell cycle.
Clinical significance.
Genetic experiments such as gene knockout (KO), which consist in modifying or inactivating a gene, can be carried out in order to see the effects of a dysfunctional SMAD 4 on the study organism. Experiments are often conducted in the house mouse ("Mus musculus").
It has been shown that, in mouse KO of "SMAD4", the granulosa cells, which secrete hormones and growth factors during the oocyte development, undergo premature luteinization and express lower levels of follicle-stimulating hormone receptors (FSHR) and higher levels of luteinizing hormone receptors (LHR). This may be due in part to impairment of bone morphogenetic protein-7 effects as BMP-7 uses the SMAD4 signaling pathway.
Deletions in the genes coding for SMAD1 and SMAD5 have also been linked to metastasic granulosa cell tumors in mice.
SMAD4, is often found mutated in many cancers. The mutation can be inherited or acquired during an individual's lifetime.
If inherited, the mutation affects both somatic cells and cells of the reproductive organs. If the "SMAD 4" mutation is acquired, it will only exist in certain somatic cells. Indeed, SMAD 4 is not synthesized by all cells. The protein is present in skin, pancreatic, colon, uterus and epithelial cells. It is also produced by fibroblasts.
The functional SMAD 4 participates in the regulation of the TGF-β signal transduction pathway, which negatively regulates growth of epithelial cells and the extracellular matrix (ECM). When the structure of SMAD 4 is altered, expression of the genes involved in cell growth is no longer regulated and cell proliferation can go on without any inhibition. The important number of cell divisions leads to the forming of tumors and then to multiploid colorectal cancer and pancreatic carcinoma. It is found inactivated in at least 50% of pancreatic cancers.
Somatic mutations found in human cancers of the MH1 domain of SMAD 4 have been shown to inhibit the DNA-binding function of this domain.
SMAD 4 is also found mutated in the autosomal dominant disease juvenile polyposis syndrome (JPS). JPS is characterized by hamartomatous polyps in the gastrointestinal (GI) tract. These polyps are usually benign, however they are at greater risk of developing gastrointestinal cancers, in particular colon cancer.
Around 60 mutations causing JPS have been identified. They have been linked to the production of a smaller SMAD 4, with missing domains that prevent the protein from binding to R-SMADS and forming heteromeric complexes.
Mutations in "SMAD4" (mostly substitutions) can cause Myhre syndrome, a rare inherited disorder characterized by mental disabilities, short stature, unusual facial features, and various bone abnormalities.


Benzyl-2-methyl-hydroxybutyrate dehydrogenase

In enzymology, a benzyl-2-methyl-hydroxybutyrate dehydrogenase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are benzyl (2R,3S)-2-methyl-3-hydroxybutanoate and NADP+, whereas its 3 products are benzyl 2-methyl-3-oxobutanoate, 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 benzyl-(2R,3S)-2-methyl-3-hydroxybutanoate:NADP+ 3-oxidoreductase. This enzyme is also called benzyl 2-methyl-3-hydroxybutyrate dehydrogenase.


Oxycholesterol

Oxycholesterol or 5,6-epoxycholesterol is a form of oxidized cholesterol implicated in atherosclerosis. It is commonly formed from the reaction of fats and oxygen during high temperature cooking such as frying.
In a study about the effects of Oxycholesterol in rabbits with induced hypercholesterolemia, it was shown that compared feeding rabbits cholesterol and unoxidized cholesterol, feeding rabbits Oxycholesterol caused there to be much higher levels of total cholesterol and LDL Cholesterol. It is also shown to increase the amount of HCY and ADMA levels in the rabbits. An abundance of HCY (Homocysteine) leads to an organism with greater chances of endothelial cell injury which can then lead to coronary artery disease. ADMA (Asymmetric dimethylarginine) is shown to cause adverse effects on humans in the form of cardiovascular and metabolic diseases. Therefore it was concluded that adding oxycholesterol into diets of animals with hypercholesterolemia increases endotheial dysfunction and inflammatory response.


Arenobufagin

Arenobufagin is a cardiotoxic bufanolide steroid secreted by the Argentine toad "Bufo arenarum". It has effects similar to digitalis, blocking the Na+/K+ pump in heart tissue.
Sources.
The compound Arenobufagin is one of the major components of toad venom( (Venenum bufonis) . It is derived from the dried skin of giant toads, such as the Bufo gargarizans or the Bufo melanostictus Suhneider. Arenobufagin is specifically secreted by the Rhinella arenarum, which is found in South America. The venom of these toad species contains about 1.75% of arenobufagin. The other major part of the venom consists mostly of similar looking bufagins, which are all toxic steroids. Toads produce their venom when they are scared, injured, or provoked, as a defense mechanism against their predators.
History.
Arenobufagin is a component of a certain toad venom which goes by the name of Chan’su. For centuries, this venom has been used in Chinese traditional medicine in the treatment of liver cancer. Also, it is reported to have a simulative effect upon the heart. It is derived from the dried skin secretions of giant toads. In a 2013 study, it was shown that arenobufagin inhibits the growth of certain liver tumor cells. In fact, this compound showed the most potent antitumor activity of fifteen bufadionolides isolated from Chan’su.
Function in medicine.
As already mentioned, arenobufagin has been widely used in traditional Chinese medicine (TCM) to tackle down carcinogenesis , since it inhibits cell growth in several cancer cells. It is one of the central active ingredients of toad venom for treatment. This happens both in purified form or in combination with other ingredients, which are mostly herbal components. Toad venom is in fact still used in clinical practice of in TCM to treat hepatocellular carcinoma (HCC). So far there is still little known about the anti-angiogenic properties of Arenobufagin. One studie shows that Arenobufagin inhibits VEGF-induced endothial cell tube formation.
Intake.
Under normal circumstances, arenobufagin is a solid. In Chinese traditional medicine, it in ingested either orally or topically, for example to the skin. Little is known about its toxicokinetics.
Toxicodynamics.
Arenobufagin is believed to play a role in the regulation of the transport of water and electrolytes across cell membranes under physiological conditions. Experimental results show that Arenobufagin inhibits the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) . It is one of the most potent blockers that are known to science, along with ouabain. It has been suggested that uncharged and non-polar amino acids may participate in the binding of arenobufagin to the extracellular surface of the ATPase. If this is the case, the steroid nucleus of arenobufagin probably also contributes to the binding. Furthermore, the presence of a sugar group in ouabain and the absence of one in arenobufagin suggests that it may me more lipophilic that ouabain and as such form a more stable complex with the Na+-K+ pump.
Besides its effects on the Na+-K+ pump, arenobufagin also has some other effects on cells, which were discovered in experiment with cancer cell lines.
It causes apoptosis by tempering with mitochondria. In cells treated with it, a decreasing mitochondrial potential was detected (in a dose-dependent manner), as well as a high Bax/Bcl-2 ratio, which is associated with apoptosis, or programmed cell death. Bax translocation from cytosol to mitochondria was also found to be increased.
In addition to these effects, arenobufagin also induces morphological changes in organelles, blebbing of plasma membrane, shrinkage of nuclear membrane and chromatin condensation. These observation also indicate the occurrence of apoptosis. Last but not least, specific cleavage of poly (ADP-ribose) polymerase (PARP) and a decrease in pro-caspase9 and 3 were also induced by arenobufagin treatment. PARP is mainly involved in cell repair and programmed cell death.
After treatment with arenobufagin, some cells make more autophagosomes and lysosomes, whereas other cells undergo apoptosis. Arenobufagin also leads to increased expression of LC3-II, Biclin1(initial vesicle formation), Atg5 (elongation and completion), Atg9, Atg16L1 and p62/SQSTM1, all proteins which induce autophagy. Blockers of autophagy increase the cytotoxic effect of arenobufagin. Addition of 3-MA increases the proportion of cells in which there in enlarged PARP cleavage and limited caspase-9 and caspase-3 cleavage. This indicates that autophagy pathways protect the cell against apoptosis by arenobufagin.
It was hypothesized that arenobufagin may inhibit the PI3K/Akt pathway in controlling cell death and differentiation in response to external stimuli. It was found that arenobufagin inhibited the proteins Akt, PDK1 and PI3K, whereas it stimulated the PHEN-protein. It does this by altering the degree of phosphorylation. All these proteins are involved in the PI3/Akt pathway. Further down the pathway, arenobufagin indirectly inhibits the mTOR-protein, which is involved in apoptosis as well as in autophagy.
Detoxification.
The biotransformation of arenobufagin by Alternaria alternata leads to the following three metabolites: 3-oxo-arenobufagin (1a), ψ-bufarenogin (1b), and 3- oxo- ψ-bufarenogin (1c). The biotransformation processes consists of a main reaction whereas the dehydrogenation of the 3-hydroxyl group takes place. This process is followed by isomerization.
Similar forms.
Arenobufagin is a derivative of the so-called bufadienolides, where it also has a steroid structure. A characteristic of bufadienolides is that they all contain two double bonds in the lactone ring.
Symptoms.
Circulatory system.
Arenobufagin works like Cardiac glycosides. It inhibits the sodium-potassium pump because it stabilises the E2-P transition state, in which the pump is inactive.
Second membrane transporter NCX is responsible for 3Na/Ca transport, if the Na-K-Pump does not function correctly the Ca concentration inside the cell will rise and this will cause heart failure.
However, in experiments concerning the anti-cancer effects of arenobufagin in mice, no negative effects where found.
Digestive system.
Arenobufagin has shown to cause appoptose in hepatocellular carcinoma cells in mice, although this method is not used to cure hepatocellular carcinoma in modern human medicine.
Structur-activity-relation.
There is a rather large homology in structure between arenobufagin and cardiac glycosides. Cardiac glycosides are natural compounds found in plants which cause inactivation of the sodium potassium pump like arenobufagin. The specific steroidic structure binds to the pump in a way it inhibits the process of pumping potassium into the cell and sodium out of the cell. The exact way of binding to the Na-K-Pump is not yet documented.
Toxicity.
Acute toxicity.
Although a low dosis of arenobufagin can be used as a medicine of heart rate problems, a high dose can lead to acute heart problems and even death. Arenobufagin is also toxic for hepatocellular carcinoma cells, which is a positive result for the body.
Chronic exposure.
Toxicity because of chronic exposure was not clearly documented thus far. However it is discussed that the chronic exposure to this compound can cause the development of tumors.


Methyclothiazide

Methyclothiazide is a thiazide diuretic.