Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LF[subscript N]) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore.MIT Start-up FundsMassachusetts Institute of Technology. Charles E. Reed Faculty Initiative FundDamon Runyon Cancer Research Foundation (Innovation Award)National Science Foundation (U.S.) (CAREER Award CHE-1351807)National Science Foundation (U.S.). Graduate Research Fellowshi

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Delivery of mirror image polypeptides into cells

Mirror image peptides have unique stability and immunogenic properties in mammals, making them attractive agents to investigate. Their properties inside cells have been mostly unexplored because biopolymers are difficult to transport across cellular membranes. Here, we used protective antigen (PA) from anthrax toxin to deliver mirror image polypeptide cargo into the cytosol of mammalian cells when conjugated to the C-terminus of the PA-binding domain of lethal factor, LF[subscript N]. We found mirror image polypeptides and proteins were translocated as efficiently into cells as their L counterparts. Once in the cytosol, by the use of western blot, we found that D peptides at the C-terminus of LF[subscript N] were able to achieve higher steady state concentrations when compared to the L-peptide conjugate. With this platform, we delivered a D-peptide MDM2 antagonist to disrupt the p53/MDM2 interaction in cancer cells. For the first time, we show the PA/LF[subscript N] system is adaptable for the intracellular delivery of mirror image peptides and proteins.Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Damon Runyon Cancer Research Foundation (Innovation Award)National Science Foundation (U.S.) (Career Award CHE-1351807)National Science Foundation (U.S.). Graduate Research Fellowship ProgramNew England Regional Center of Excellence for Biodefense and Emerging Infectious DiseasesMassachusetts Institute of Technology. Biophysical Instrumentation Facilit

Hepatoma cell uptake of cationic multifluorescent quantum dot liposomes

Cationic multifluorescent quantum dot liposomes (QD-Ls) have been prepared with both hydrophobic and hydrophilic CdSe/ZnS quantum dots by reverse phase evaporation. QD incorporation was confirmed by fluorescence and confocal microscopy. Incorporation did not affect QD photoactivity or damage bilayer or liposome structure. Cell uptake was examined in human hepatocellular carcinoma cells (HuH-7) using cationic and zwitterionic QD-Ls. Cationic QD-Ls were stable in vitro and exhibited high uptake, while zwitterionic QD-Ls aggregated and exhibited low uptake. Given that liposomes are established and versatile platforms for creating cell-targeting therapeutic agents, multifluorescent QD-Ls may offer advanced techniques for imaging hydrophobic and hydrophilic domains simultaneously. If coupled with an encapsulated drug, QD-Ls could be multifunctional and provide imaging, detection, and drug delivery in a single assembly. © 2009 American Chemical Society

Protein Thioester Synthesis Enabled by Sortase

Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal ^αthioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an ^αthioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation

Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase

Ras/Rap1-specific endopeptidase (RRSP) is a cytotoxic effector domain of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of highly virulent strains of <i>Vibrio vulnificus</i>. RRSP blocks RAS-MAPK kinase signaling by cleaving Ras and Rap1 within the switch I region between Y32 and D33. Although the RRSP processing site is highly conserved among small GTPases, only Ras and Rap1 have been identified as proteolytic substrates. Here we report that residues Y32 and D33 at the scissile bond play an important role in RRSP substrate recognition, while the nucleotide state of Ras has an only minimal effect. In addition, substrate specificity is generated by residues across the entire switch I region. Indeed, swapping the Ras switch I region into either RalA or RhoA, GTPases that are not recognized by RRSP, generated chimeras that are substrates of RRSP. However, a difference in the processing efficiency of Ras switch I in the context of Ras, RalA, or RhoA indicates that protein regions outside Ras switch I also contribute to efficient RRSP substrate recognition. Moreover, we show that synthetic peptides corresponding to the Ras and Rap1, but not RalA, switch I regions are cleaved by RRSP, demonstrating sequence-specific substrate recognition. In conclusion, this work demonstrates that the GTPase recognition of RRSP is independent of the nucleotide state and is mainly driven by the Ras and Rap1 switch I loop and also influenced by additional protein–protein interactions, increasing the substrate specificity of RRSP

Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids

Copyright © 2020 American Chemical Society. Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer