Mushroom Amanitine

One peptide toxin from the mushroom Amanita phalhides, a-amanitin, is a specific differential inhibitor of the eukaryotic nuclear DNA-dependent RNA polymerases and as such has proved to be a powerful research tool (Table 37-2). a-Amanitin blocks the translocation of RNA polymerase during transcription. 

Mushroom-Aman/fa pf7a//o/des Amanitins - peptide-based toxins 200... 

Transcription factors (such as TFIID for RNA polymerase II) help to initiate transcription. The requirements for termination of transcription in eukaryotes are not well understood. All transcription can be inhibited by actinomycin D. In addition, RNA polymerase II is inhibited by a-amanitin (a toxin from certain mushrooms). These points are summarized in Table 1-3-1,... 

Inhibited by rifampin Actinomycin D RNAP 2 inhibited by a-amanitin (mushrooms) Actinomycin D... 

Amatoxins are a family of cyclic peptides, with oc-amanitin and 3-amanitin (Figure 3.1) accounting for >90% of the total amatoxins. In A. virosa, mushrooms collected in Virginia a-amanitin found to be completely replaced by amaninamide (Figure 3.1). The peptides are not destroyed by cooking and... 

Saviuc, P., Dematteis, M., Mezin, P., Danel, V. and Mallaret, M. (2003). Toxicity of the Clitocybe amoenolens mushroom in rat, Vet. Hum. Toxicol, 45, 180-182. Seeger, R. (1984). Zeitungspapiertest fiir Amanitine - falsch-positive Ergebnisse, Z. 

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

This mushroom produces the toxin, a-amanitin, a cyclic octapeptide having several modified amino acids and a central purine, which strongly binds to and inhibits RNA pal ii and thereby blocirs elongation. 

Many small peptides exert their effects at very low concentrations. For example, a number of vertebrate hormones (Chapter 23) are small peptides. These include oxytocin (nine amino acid residues), which is secreted by the posterior pituitary and stimulates uterine contractions bradykinin (nine residues), which inhibits inflammation of tissues and thyrotropin-releasing factor (three residues), which is formed in the hypothalamus and stimulates the release of another hormone, thyrotropin, from the anterior pituitary gland. Some extremely toxic mushroom poisons, such as amanitin, are also small peptides, as are many antibiotics. 

The mushroom Amanita phalloides has evolved a very effective defense mechanism against predators. It produces a-amanitin, which disrupts mRNA formation in animal cells by blocking Pol II and, at higher concentrations, Pol III. Neither Pol I nor bacterial RNA polymerase is sensitive to a-amanitin—nor is the RNA polymerase II of A. phalloides itself ... 

Amanitin. Toxin from poisonous mushroom Aminita phalloides... 

The eukaryotic RNA polymerases are not inhibited by rifamycin, but RNA polymerases II and III are completely inhibited by the mushroom poison a-amanitin (see Box 28-B). Inhibitors of DNA gyrase (Chapter 27) also interfere with transcription as do chain terminators such as cordycepin (3 -deoxyadenosine) and related nucleosides. 

The same mushrooms contain several fast-acting toxic heptapeptides, the phalloidins, whose structures are similar to those of the amanitins. However, they contain a reduced sulfur atom (—S—) in the cross-bridge. They are specifically toxic to the liver.c The same mushrooms also contain an antidote to the phalloidins, antamanide. This cyclic... 

Nuclear extracts can be fractionated by chromatography on DEAE-cellulose to give three peaks of RNA polymerase activity (the use of column chromatography is explained in chapter 6). These three peaks correspond to three different RNA polymerases (I, II, and III), which differ in relative amount, cellular location, type of RNA synthesized, subunit structure, response to salt and divalent cation concentrations, and sensitivity to the mushroom-derived toxin a-amanitin. The three polymerases and some of their properties are summarized in table 28.4. 

The most useful inhibitor of eukaryotic transcription has been a-amanitin, a major toxic substance in the poisonous mushroom Amanita phalloides. The toxin preferentially binds to and inhibits RNA polymerase II (see table 28.4). At high concentrations it also can inhibit RNA polymerase III but not RNA polymerase I or bacterial, mitochondrial, or chloroplast RNA polymerases. 

Another class of toxins produced by some mushrooms consists of polypeptides, particularly amanitin and phalloidin. These substances are stable to heating (cooking). They are systemic poisons that attack cells of various organs, including the heart and liver. In early 1988, an organ transplant was performed on a woman in the U.S. to replace her liver, which was badly damaged from the ingestion of wild mushrooms that she and a companion had mistakenly identified as edible varieties and consumed. 

Eukaryotic cells contain at least four different DNA-dependent RNA polymerases. Their localization, cellular transcripts, and susceptibility to the cyclic octapeptide a-amanitin (derived from poisonous mushrooms) are shown in Table 11.3. a-Amanitin blocks the elongation phase of RNA synthesis. Although the structures of these enzymes are much more complex than that of the prokaryotic RNA polymerase, the basic mechanism is very similar to that of the prokaryotic enzyme. 

No. Eukaryotic RNA polymerases have been isolated from many tissues, and in all cases, three distinct enzymes have been found in the nucleus. All contain a number of polypeptide subunits and are complex in structure, RNA polymerase I is known to be involved specifically in the transcription of rRNA genes. RNA polymerase II gives rise to transcripts that are subsequently processed to yield mRNA. RNA polymerase 111 is responsible for the transcription of the tRNA genes and a small ribosomal RNA gene that yields a species called 55 RNA. The three polymerases are distinguishable from one another by their differential sensitivity to the drug a-amanitin (the toxic principle of the mushroom Amanita phalloides), which does not affect bacterial RNA polymerase. RNA polymerase... 

Maurer, H.H., Schmidt, C.J., Weber, A.A., Kraemer, T. Validated electrospray LC MS assay for determination of the mushroom toxins alpha- and beta-amanitin in urine after immunoaffinity extraction. J. Chromatogr. B Biomed. Sci. Appl. 748, 125-135 (2000)... 

Figure 28.16. RNA Polymerase Poison. Amanita phalloides, a poisonous mushroom that produces a-amanitin. [After G. Lincoff and D. H. Mitchel, Toxic and Hallucinogenic Mushroom Poisoning (Van Nostrand Reinhold, 1977), p. 30.]...

The mode of action of mushroom-produced mycotoxins varies considerably. Alpha amanitin, amatoxin produced by some species of Amanita, is a class A poison that acts by inhibiting a critical nuclear polymerase that enables the cell to make protein. Once the function of this RNA polymerase is curtailed, basic life processes cease. Attempts to kill alpha amanitin with antibodies have proven to be even more harmful to patients than the poison itself. Most forms of mushroom poisoning can be treated with rapid lavage (induced vomiting) or medically approved ingestion of charcoal to absorb the toxin before it is absorbed into the stomach. 

Eukaryotes have four different RNA polymerases (RNA pol). Three are required for transcription of nuclear genes and the fourth for transcription of mitochondrial genes (Fig. 11.6). RNA polymerase I transcribes ribosomal RNA (rRNA), pol II transcribes mRNA and pol III tRNA and several small RNA s. The three polymerases consist of ten or more subunits. All have two large subunits with homology to the P and p subunits of the prokaryotic RNA polymerase. The three eukaryotic polymerases can be distinguished based on their sensitivity to a-amanitin, a toxin found in some types of mushrooms. RNA pol II activity is severely inhibited, pol III weakly and pol I is insensitive. The antibiotic rifampicin inhibits prokaryotic RNA polymerases. 

It also should be noted that within the genus Amanita there exist several species of deadly mushrooms. They are among the small number of mushroom species the ingestion of which can prove fatal. These species include Amanita phalloides and Amanita viA, both of which contain small peptides called amanitins that inactivate RNA polymerase and cause irreversible damage to liver function. 

B. The poison in poisonous mushrooms is a-amanitin, an inhibitor of eukaryotic RNA polymerases. 

Gundala, S., Wells, L.D., Milliano, M.T., Talkad, V., Luxon, B.A. and Neuschwander-Tetri, B.A. (2004) The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin alpha-amanitin. Archives of Toxicology, 78, 68-73. 

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