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.