No description available.

Ribosome-inactivating proteins (RIPs) such as Shiga-like toxin 1 (SLT-1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin-ricin loop of 28S rRNA. The molecular details involved in the ER lumenal escape and subsequent site-specific depurination are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Using a combination of yeast-2-hybrid and HeLa lysate pull-down followed by LC-MS/MS we have discovered yeast and human proteins that interact with the catalytic A1 chain of SLT-1. Yeast-2-hybrid library screens followed by the expression of full-length protein candidates and pull-down experiments yielded Cue2 as the only yeast cellular component that binds to the SLT-1 A1 chain. Further truncational analysis revealed that the known protein domains (two Cue domains and a Smr domain) within the primary sequence of Cue 2 were not essential for the interaction. Cue2 is a yeast monoubiquitin binding protein of no known function that is structurally homologous to the human ubiquitin-associated domain which has been implicated in intracellular routing and ER-associated degradation. Pull-down experiments indicated that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved acidic C-terminal peptide domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT-1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, phage-display was used to rapidly probe the importance of each residue within this C-terminal ribosomal peptide. The analysis revealed a complementary acidic surface and an additional hydrophobic motif involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT-1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.

Ribosome-inactivating proteins (RIPs) such as Shiga-like toxin 1 (SLT-1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin-ricin loop of 28S rRNA. The molecular details involved in the ER lumenal escape and subsequent site-specific depurination are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Using a combination of yeast-2-hybrid and HeLa lysate pull-down followed by LC-MS/MS we have discovered yeast and human proteins that interact with the catalytic A1 chain of SLT-1. Yeast-2-hybrid library screens followed by the expression of full-length protein candidates and pull-down experiments yielded Cue2 as the only yeast cellular component that binds to the SLT-1 A1 chain. Further truncational analysis revealed that the known protein domains (two Cue domains and a Smr domain) within the primary sequence of Cue 2 were not essential for the interaction. Cue2 is a yeast monoubiquitin binding protein of no known function that is structurally homologous to the human ubiquitin-associated domain which has been implicated in intracellular routing and ER-associated degradation. Pull-down experiments indicated that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved acidic C-terminal peptide domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT-1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, phage-display was used to rapidly probe the importance of each residue within this C-terminal ribosomal peptide. The analysis revealed a complementary acidic surface and an additional hydrophobic motif involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT-1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

No description available.

Proteins with selective toxicity have been investigated for use in many ways. One class of proteins, ribosome-inactivating proteins (RIPs), is found throughout the plant kingdom as well as in lower organisms like certain fungi and bacteria. These are a group of proteins that has the property of damaging the ribosomes in an irreversible manner. They are N-glycosidases that modify the 28S rRNAs to render them incapable of sustaining further translation. RIPs have been divided into two groups, i.e. type I RIPs, which are single polypeptide chains and type II RIPs, which are heterodimeric. Abrin is a type II RIP, isolated from the seeds of Abrus precatorius plant commonly known as jequirity plant. It is a heterodimeric glycoprotein consisting of an A and a B subunit linked together by a single disulfide bond. The toxicity of the protein comes from the A subunit harboring the RNA-N- glycosidase activity which catalyses the depurination of a specific adenine residue at position 4324 on the 28S rRNA. The depurination of the adenine prevents the formation of a critical stem loop structure to which the elongation factor -2 (EF-2) binds during the translocation step of the translation, thus stalling the translation machinery of the cells. The B subunit of abrin is a galactose specific lectin. The lectin activity enables the protein toxin to bind to the cell surface glycoproteins and/or glycolipids. Binding of abrin is followed by internalization of the protein by receptor mediated endocytosis and transport to the Endoplasmic reticulum (ER) by the retrograde transport pathway. Inside the ER, the single disulfide bond linking the two subunits, is reduced which is important for the A subunit toxicity. The A subunit then translocates into the cytosol using the ER-associated degradation (ERAD) pathway and cleaves the specific adenine residue on the 28S rRNA of the 60 S ribosome involved in active translation and thereby inhibiting the protein synthesis. In addition to its ability to inhibit translation, abrin induces apoptosis in cells. Earlier work from our laboratory has shown that abrin-induced apoptosis follows the intrinsic pathway of apoptotic cell death. The treated cells show mitochondrial membrane potential loss followed by caspases -9 and -3 activation and DNA fragmentation. RIPs have been used primarily in immunotherapy because of their toxicity at very low concentrations (picomolar). With the development of monoclonal antibodies as tool for targeting cell surface markers, the possibility to couple antibodies to RIPs and thus deliver the toxic protein directly to specific cells becomes feasible. Abrin, as one such potent RIP, has gained interest in the field of medicine and immunotherapeutics. Abrin can also be a candidate for use in bioterrorism and warfare. Therefore, it is very important to first understand the inhibitory effect of abrin and the extent of its toxicity on cells. Earlier studies from our laboratory have focused on the sensitivity and mechanism of cell death induced by abrin in Jurkat cells, a T –cell line. In the present study, we attempted to investigate the overall toxicity of the molecule with respect to both properties, inhibition of protein translation and induction of apoptosis, in different lineages of cells. We have carried out a comparative study on abrin toxicity on human cell lines from two different cell lineages namely hematopoietic and epithelial. The thesis is divided into introduction and two chapters. In the introduction, we have presented the general properties of this family of proteins, with a brief history; classification and distribution of plant RIPs and their enzymatic properties. The chapter also deals with possible usage of these proteins, mainly in the field of immunotherapy. We have introduced, abrin, the protein of our interest in this chapter. The structure of abrin is described and also the biological effects of the toxin are discussed in brief. The chapter one deals with the translation inhibitory property of the protein, abrin. As mentioned earlier, abrin inhibits protein synthesis via the RNA-N-glycosidase activity residing in its A-chain. We have presented the general cytotoxic pathway of type II RIPs in this chapter. It deals with the internalization and transport of the toxin to their site of action, the cytosol. As reported earlier, our results confirmed that abrin inhibited protein synthesis in all cells. Abrin mediated inhibition of translation was dose dependent. Though the inhibition was common to all the cells from both the lineages, the sensitivity of the cells towards the toxin and kinetics of this inhibition event differed significantly. The kinetics of inhibition of protein synthesis is faster in case of hematopoietic cells as compared to the epithelial cells even at lower doses of the toxin. These differences were not due to variations in the ability of protein synthesis of cells. The chapter also discusses binding of the protein to cells. Our data suggest that binding of abrin to the cells is not responsible for the variations observed in the translation inhibitory property of the protein except in Raji cells. The B-cell line Raji was found to be least sensitive towards the toxin. Our studies show that due to presence of high sialic acid residues on the surface of these cells, Raji cells are refractory to abrin mediated inhibition of protein synthesis. The second chapter presents our data on cell death upon abrin treatment. This part is divided into an introduction and two sections, A and B. In the introduction, different cell death modalities are discussed along with recent findings in the field of programmed cell death. Section A deals with abrin induced apoptosis in epithelial cells. We have compared the extent of abrin-triggered apoptosis in these cells. Some of the early events known in the apoptotic cascade of abrin are compared. Though apoptosis is observed in these cells, our data suggest a delay in the apoptotic trigger in the epithelial cells showing that epithelial cells can survive the stress induced by abrin for a longer time. When treated with other apoptotic agents, like etoposide, these cells are found to be resistant. Therefore, though there is a delay in the trigger of apoptosis, we have shown that the cells tested from the epithelial lineage undergo apoptosis on abrin treatment. Section B, discusses the ability of the protein to induce cell death in hematopoietic cells. We have presented studies on cell death other than apoptosis, detected in these cells upon abrin treatment. We found that some of the cell lines tested undergoes more necrosis than apoptosis with abrin treatment. When the status of the mitochondria was checked, we found that in U266B1 cells, a B-cell line, there was mitochondrial stress as well as reactive oxygen species (ROS) production. But these cells died by necrosis. The data obtained from this study show the involvement of lysosomes and cathepsins in abrin induced cell death in U266B1 cells. Though other cells also undergo necrosis, these events were unique to U266B1 cells.