Therapeutic Application of Genetically Engineered Ribosome-Inactivating Toxin Proteins for Cancer

Recently, Ribosome-Inactivating Proteins (RIPs) as a class of anticancer medicines have garnered considerable attention due to their novel anticancer mechanism. Although the medications are small, RIPs utilize the Large-Size Effect (LSE) to block the efflux procedure that are regulated through Drug Resistance Transporters (DRTs), and protect host cells from drug transfection. There are many significant challenges for their therapeutic applications that seriously restrict their usefulness, particularly their strategy towards tumor cells. The primary objective of this review is to emphasize Trichosanthin (TCS) along with Gelonin (Gel) and additional types of RIPs, particularly scorpion venom-derived RIPs, to demonstrate that they should be grappling through what kinds of bio-barriers to overcome in cancer therapeutic science. Next, we will emphasize the latest state-of-the-art in providing cancer treatment RIPs.

Containment of Phytoplasma-Associated Plant Diseases by Antibiotics and Other Antimicrobial Molecules

Phytoplasmas are plant-pathogenic bacteria that infect many important crops and environmentally relevant plant species, causing serious economic and environmental losses worldwide. These bacteria, lacking a cell wall, are sensitive to antibiotics such as tetracyclines that affect protein synthesis mechanisms. Phytoplasma cultivation in axenic media has not been achieved for many strains; thus, the screening of antimicrobials must be performed using mainly in vivo materials. Some studies have investigated using in vitro phytoplasma-infected shoots, and several antimicrobials, including tetracyclines, have been tested. The screening of phytoplasma antimicrobials is important for the sustainable control of phytoplasma-associated diseases. The use of molecules with different modes of action such as ribosome inactivating proteins, plant hormones, and resistance inducers such as plasma-activated water, is advised, to avoid the use of antibiotics in agriculture and the possible emergence of resistant microbial strains.

Lyophyllin, a Mushroom Protein from the Peptidase M35 Superfamily Is an RNA N-Glycosidase

Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from Lyophyllum shimeji, an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients’ gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells’ growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

Using Oxford Nanopore Technology direct RNA sequencing to identify depurination events induced by ricin and other ribosome inactivating proteins

Depurination is a frequent modification to both DNA and RNA, in DNA causing point mutations through misincorporation, in RNA, disabling ribosomes and halting protein synthesis. Some modifications of nucleic acids can be determined by direct sequencing using Oxford Nanopore Technologies (ONT). However, the identification of modifications is often limited by noise and their variety and number. Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop of the large ribosomal subunit. This leaves only a ribose backbone, thus inhibiting protein translation. In humans, biological threat agents and ribosome inactivating proteins, such as ricin and saporin, depurinate base 4605 on the 28S rRNA providing a single defined target to try and identify. We postulated that the depurination event could be detected using ONT direct RNA sequencing through a change in charge in the ricin loop. A software tool was developed, RIPpore, that quantified the adenine modification from direct RNA sequencing data of ribosomal RNA purified from respiratory epithelial cells exposed to ricin. This provided a novel method of directly identifying ricin exposure and a basis for the utility of ONT in detecting lesions in nucleic acids caused by depurination events.

Molecular aspects of creating vaccines for the prevention of poisoning ribosome-inactivating proteins of plant origin: current situation, problems of vaccine development

This article reviews the current understanding of the mechanism of action of the toxin, the clinical effects of ricin and abrin intoxication and how these relate to current and continuing prospects for vaccine development. The threat of bioterrorism worldwide has accelerated the demand for the development of therapies and vaccines against the ribosome-inactivating proteins. The diverse and unique nature of these toxins poses a challenge to vaccinologists. This paper will review the mechanism of toxicity and vaccines development to protect against the highly toxic plant-derived ribosomal toxins. Vaccine development is further complicated by the fact that as bioterrorism agents, abrin and ricin would most likely be disseminated as aerosols supplies. Our understanding of the mechanisms by which these toxins cross mucosal surfaces, and importance of mucosal immunity in preventing toxin uptake is only rudimentary. Research is now aimed at developing recombinant, attenuated vaccines based on a detailed understanding of the molecular mechanisms by which these toxins function. The evolution of the development of specific immunoprophylaxis of acute ricin poisoning from native toxoid to genetically engineered subunit vaccines based on the method of targeted mutagenesis is traced. The past several years have seen major advances in the development of a safe and efficacious ricin toxin vaccine. These vaccines are discussed in the context of the toxicity and structure of ricin. In this review we summarize ongoing efforts to leverage recent advances in the design and use of vaccines.

Novel Ribosome-inactivating Protein (RIP) Isolated from Trichosanthes dioica Induces Apoptosis in HeLa Cell Line

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate eukaryotic and prokaryotic rRNAs and thus interrupt protein synthesis during translation. In the present study, a protein of around 32 kDa, supposedly a RIP isolated from Trichosanthes dioica, was assessed for its potential to induce apoptosis in HeLa cells. Cell viability assay was done to measure cell proliferation and survivability. It was observed that cells viability decreased with the increase of decrease in dilution, i.e. when the sample was an undiluted one, the viability decreased drastically and almost came to less than 10%. To further check whether the isolated RIP could induce apoptosis, HeLa cells were treated with the test RIP. Immunoblotting was carried out using PARP poly (ADP-ribose) polymerase (PARP-1), a 113 kDa nuclear enzyme, which is considered a hallmark of cells undergoing apoptosis. HeLa cells were further analyzed for loss of mitochondrial membrane potential with JC-1 dye, which is an early event during apoptosis. Increased PARP breakdown in the RIP treated cells indicates that cells undergoing apoptosis and progressive loss of red J-aggregate fluorescence indicate that the isolated RIP from Trichosanthes dioica induces apoptosis in HeLa cells. The ability of apoptosis induction is comparable to another known RIP from Momordica charantia, which was used as a positive control. Promising results from the present study warrants the isolated RIP to be further explored for anticancer activities.