Ribosome-inactivating proteins (RIPs) are a class of toxic proteins that catalyze the ribosome r RNA depurination reaction, thereby inhibiting ribosomal function.
The main representatives are ricin (ricin) and acacia toxin (abrin), which are important biological warfare agents and bioterrorism weapons, and there is an urgent need to study detection methods and inhibitors of such toxins to ensure public safety and poisoning treatment.
Due to the N-glycosidase activity of these toxins, depurine reactions can occur on certain short-stranded DNA or RNA, and instrumental analysis can be used to quantitatively detect the released adenine or the oligonucleotide single strands after depurination.
Based on this principle, a total of 29 whole RNA sequences and DNA-RNA-DNA chimeric sequences were designed, and their activities were evaluated, and a total of 8 new effective nucleic acid substrates Risub4, Risub12, Risub13, Risub14, Risub15, Risub17, Risub18, Risub19, Risub20, Risub21 and rA12d6 were screened.
Through the determination and comparison of enzyme kinetic parameters, rA12d6 was found to be the most active oligonucleotide substrate, and the activity detection and quantitative detection method of ricin was established in combination with the high-performance liquid phase, with a minimum detection concentration of 0.1 ng/mL and a detection limit LOD value of 2.3 ng/mL.
Secondly, the functional base conservation of ricin nucleic acid substrates and the design and synthesis of nucleic acid inhibitors were carried out. Using the stem ring structure dA12 formed by 12 nucleotide units (12-nt) as a template, 28 nucleic acid sequences were synthesized.
Nucleoside analogues 1-4 were designed for the 6-position amino group and 7-position nitrogen atom of deoxygenated adenosine in the center of the ring and the 7-position nitrogen atom of deoxyguanosine, and the modified nucleic acid sequences RIN10-RIN12 and RIN16-RIN28 were synthesized, and it was found that the change of any functional group on the four-member cyclic base would affect the function of dA12 as a substrate.
On the other hand, the 6-position amino group and 8-position of deoxyadenosine in the center of the ring introduced benzyl groups with planar stacking ability and aminopropyl and histamine groups with strong hydrogen-forming bonding ability, nucleoside analogues 5-7 were designed, and the modified nucleic acid sequences RIN01-RIN09 and RIN13-RIN15 were synthesized, and after the evaluation of inhibitory activity, four nucleic acid sequences with obvious inhibitory activity RIN06, RIN11, RIN16 and RIN18 were screened, and the inhibition reached the micromolar level.
This provides further guidance for the inhibitor design and chemical modification methods of type II RIPs.
Ribosome inactivating proteins and their classification
From the current research on toxin proteins such as ribosomal inactivation protein, ricin, acacia toxin, saponatotoxins, and Shiga toxin have attracted the most attention. In particular, ricin and acacia toxins have strong toxicity, wide sources, cheap and easy availability, stable properties, and no antidote, and have become important biochemical warfare agents and bioterrorism weapons.
The 1978 Markov political murder used ricin. Moreover, ricin mailings targeting important political figures have been reported from time to time. As such, they are listed in Schedule 1A of the Chemical Weapons Convention (CWC) for monitoring and are Category B substances under the Biological and Toxin Weapons Convention (BTWC).
According to the functional domain composition, ribosomal inactivating proteins can be roughly divided into three categories, namely single-stranded type I RIPs, double-stranded type II. RIPs, and multi-stranded type II.I RIPs.
Type I RIPs are a class of N-glycosidase proteins with a single structure that cannot enter cells, so type I RIPs are generally less toxic. Type II RIPs are composed of A chain with N-glycosidase activity (the same function as type I RIPs) and lectin B chain, which can bind to receptors on the surface of the cell membrane such as galactose receptors, and mediate the entry of A chain into the cell.
In addition, there are a third type of RIPs, but only a few proteins are included, highlighted by jasmonate-inducible protein (JIP60) and corn b-32 protein.
In 2010, a new nomenclature was proposed, corresponding to the traditional classification methods: type A (type I), type AB (type II.), type AC (type II.I, similar to JIP60) and AD type (type II.I, similar to maize b32 protein).
Type II ribosome inactivating protein toxicity and mechanism of action
Type II ribosome inactivating protein toxicity
Among the three types of RIPs toxins, type II RIPs have the strongest toxicity and the most poisoning cases, and their toxicity manifestations are very different, which may be related to endocytic pathways and cell types.
RIPs utilize different endocytic pathways during cell entry and bind to a variety of molecules. On the other hand, different types of cell surface distribution have different ligands and abundances, so the endocytosis of RIPs is different, thus showing differences in toxicity.
Among type II RIPs, ricin and acacia toxin are typically represented. Ricin can inactivate thousands of ribosomes in 1 minute, and the LD50 value of intravenous injection of mice is 2.7 μg/kg, which will damage the liver, kidney and other substantial organs, and cause bleeding, degeneration, and necrotic lesions. The lethal dose to humans varies depending on the route of ingestion, with an LD50 value of approximately 3 μg/kg.
Acacia toxin is approximately 75 times more toxic than ricin, and the lethal dose ingested by adults is estimated to be 0.1-1.0 μg/kg. Other type II RIPs have different toxicity, such as Shiga toxin. is also highly toxic.
SNLRP1 extracted from European elderberry is a non-toxic type II RIPs, and Nigrin b, also derived from European elderberry, not only has no lectin properties, but is also non-toxic because it can be rapidly broken down and excreted by cells.
Although type I RIPs have difficulty entering cells due to the lack of B chains, they exhibit strong toxicity if they can be connected to appropriate vectors and gain access to cells. The first proof of this is the binding of white tree toxin to canavalin, which is more cytotoxic than type II RIPs.
At present, there are many studies on the poisoning mechanism of ricin, first of all, its toxicity has obvious dose-dependence, mainly manifested as inhibition of protein synthesis at high concentrations leading to cell necrosis, and low concentrations causing lipid peroxidative damage and inducing apoptosis.
It is worth noting that ricin and other RIPs not only cleave adenine at specific sites of rRNA, but also have adenine activity against DNA, indicating its extensive disruption of nucleic acid structures and thus wider toxicity.
Second, these toxins have the ability to induce cytokines that can explain multiple cell death pathways caused by ricin and other RIPs, including apoptosis and necrosis.
At present, for the poisoning treatment of such biotoxins, because there is no antidote, clinical only supportive therapy can be used to minimize the poisoning effect, such as gastric lavage, excretion, dialysis and other methods.
Mechanism of type II ribosome inactivation protein
Based on the strong toxicity of ricin and the actual poisoning and poisoning events, therefore, the poisoning mechanism, catalytic reaction mechanism, and antitoxic drug research of ricin have attracted the most attention.
Ricin is formed by the ligation of two functional proteins by disulfide bonds, one of which is the A chain (RTA) of N-glycosidase activity, which performs a depurine reaction on ribosomal rRNA; The second is the lectin protein B chain (RTB), which has the function of endophasing cells.
First, RTB binds to N-acetylgalactose receptors on glycolipids and glycoproteins on the cell membrane surface and enters the cell through receptor mediation. In addition, it can enter the cytoplasm through mannose receptors on the surface of macrophages and hepatic sinus cells.
After entering the cytoplasm by endocytosis, ricin is transported to the early endosome, where most of the toxin molecules are recycled to the cell surface, or transported to the lysosome for degradation through the late endosome, which has an acidic environment, similar to lysosomes, and facilitates the dissociation of some toxin-receptor complexes, and the dissociated receptors can return to the surface of the cell membrane to re-function.
A small part of the toxin first enters the endoplasmic reticulum through the reverse transport system with the help of Golgi apparatus, at this time, under the action of protein disulfide isomerase in the endoplasmic reticulum, the two chains of A/B are dissociated, and the A chain enters the cytoplasm and refolds into an active conformation, exerting N-glycosidase activity and producing cytotoxicity.
In the cytoplasm, RTA acts on the A4324 position on the ribosomal 60S large subunit 28S rRNA, and a depurine reaction occurs. A4324 is located in a conserved circular structure sarcin-ricin RNA loop (SRL) in 28S rRNA.
SRL consists of a double helix stem and a 17-base ring with a conserved fragment GAGA in the center, where the second adenine is the A4324 position of the 28S rRNA.
After the depurine reaction, the aminoacyl tRNA-elongation factor EF1-GTP ternary complex cannot bind to the ribosome A position, blocking the amino acid carry process, and also interferes with the formation of the elongation factor EF2-ribosomal-GTP ternary complex, hinders the translocation of the primary protein chain from A to P on the ribosome, irreversibly inhibits protein synthesis, and leads to cell death.
The stem-loop high-level structure of the depurine site on the ribosome consists of four conserved bases that are required for the depurination reaction of ricin. For this reason, a variety of truncated nucleic acid stem ring substrate models were designed, represented by rA12N.
Ricin has a depurine half-life of 8 minutes for r A12N, which can be studied as an alternative to natural substrates. Detailed structural studies have shown that GAGA fragments are key domains in annular regions, and Watson-Crick pairing of double helix stem structures is also necessary.
However, the recognition of enzymes and substrates is not very dependent on the specific sequence of the stem structure. This truncated stem cyclic nucleic acid sequence was later used as a structural template for RIPs inhibitor research, opening up new inhibitor design ideas.
For the catalytic depurination reaction mechanism of ricin and acacia toxin, amino acid residue mutation and crystal structure analysis were mainly used.
Studies have shown that their catalytic domain composition is very similar, although the primary amino acid sequences are not the same, but the spatial location of key catalytic amino acids is similar.
A slight change in its position did not affect the depurination reaction of the nucleic acid substrate. Based on the crystal structure analysis of ricin and the effect of key amino acid mutations on catalytic activity, as well as the crystal structure analysis of ricin and a variety of substrate analogues or small molecule inhibitors, an in-depth understanding of the catalytic domain and catalytic mechanism of ricin has been obtained.
In the crystal structure of the methinomycin phosphate (FMP)-RTA complex, FMP acts as an analogue of AMP (adenosine phosphate) whose heterocyclic plane forms a π-π stacking with Tyr80 and Tyr123 of RTA.
On the other hand, mutations in the above amino acids involved in the interaction also confirmed their effect on substrate recognition.
If Tyr80 and Tyr123 are mutated to Phe, respectively, the kcat of RTA is reduced by 15 times and 7 times, respectively. When they mutate to Ser, respectively, they result in a 160- and 70-fold decrease in activity, respectively.
From this mutation study, it is shown that the benzene ring of Tyr80/Tyr123 is a key functional group, and a hydrophobic pocket is constructed with its planar benzene ring, while the hydroxyl group on the ring does not affect the catalytic reaction itself, and it is speculated that its possible hydrogen bond between the surrounding groups has a certain effect on the local conformation of the catalytic domain, so that the catalytic reaction group is in a more favorable position.
Thus, Tyr80 and Tyr123 play a key role in the affinity and specificity of substrate binding events.
In addition, FMP also forms multiple hydrogen bonds with surrounding amino acids such as Gly121, Arg80 and Val81, which indicates that the functional groups and spatial magnitude between the substrate and the RTA catalytic domain are matched, which further strengthens the recognition specificity of RTA for adenine bases.
Combined with the above analysis, the depurine reaction mechanism of RTA on ribosome substrates is proposed. Tyr80 and Tyr23 of RTA are located deep in the slit at the reaction site, with which the first adenosine unit (depurine site) of the substrate fragment GAGA forms a π-π stack.
Two other key amino acids, Glu177 and Arg180, were involved in the catalytic reaction of RTA. They have the ability to transfer protons (Figures 1-3), activate glycosidic bonds of water molecules and adenosine units, and stabilize the formation of transition states.
Arg180 is thought to protonate the N3 of adenine, followed by the transfer of electrons on the ring, resulting in cleavage of C1'-N9. At C1' an oxygen-carbod is produced, stabilized by the water molecule activated by Glu177.
When Glu177 or Arg180 is converted to Gln, it results in a 1000-fold and 200-fold decrease in activity, respectively. Moreover, the mutations of Glu177 and Arg180 have little effect on the Km of RTA, which also indicates that they are catalytic functional groups.
There is also a conserved Trp211 in the catalytic domain of RTA, and its contribution to catalytic reactions needs to be further studied.
In spatial location, it is parallel to Arg211 and may contribute to the formation of catalytic domains. Consistent with this hypothesis, when Trp211 mutates to Phe, the effect on activity is minimal.