RAS specific protease induces irreversible growth arrest via p27 in several KRAS mutant colorectal cancer cell lines.

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.

The respiratory syncytial virus (RSV) prefusion F-protein functional antibody repertoire in adult healthy donors.

Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre-existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell-sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F-protein (cross-binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross-binder nAbs used a wide array of gene rearrangements, while preF-binder nAbs derived mostly from the expansion of B-cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F-protein, identifying an important site of pathogen vulnerability.

Convergent structural features of respiratory syncytial virus neutralizing antibodies and plasticity of the site V epitope on prefusion F.

Respiratory syncytial virus (RSV) is a global public health burden for which no licensed vaccine exists. To aid vaccine development via increased understanding of the protective antibody response to RSV prefusion glycoprotein F (PreF), we performed structural and functional studies using the human neutralizing antibody (nAb) RSB1. The crystal structure of PreF complexed with RSB1 reveals a conformational, pre-fusion specific site V epitope with a unique cross-protomer binding mechanism. We identify shared structural features between nAbs RSB1 and CR9501, elucidating for the first time how diverse germlines obtained from different subjects can develop convergent molecular mechanisms for recognition of the same PreF site of vulnerability. Importantly, RSB1-like nAbs were induced upon immunization with PreF in naturally-primed cattle. Together, this work reveals new details underlying the immunogenicity of site V and further supports PreF-based vaccine development efforts.

An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth.

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.

Anthrax Protective Antigen Retargeted with Single-Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells.

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LFN ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.

Direct Cloning Method for Expression of Recombinant Proteins with an Inositol Hexakisphosphate Inducible Self-Cleaving Tag.

Protein purification is the most basic and critical step for protein biophysical and biochemical studies to understand its function and structure. Various fusion tags and proteases have been developed and assembled in expression and purification system. However, it is one of the fields that continues to innovate to develop improved systems that are more efficient, simpler, and less expensive. An efficient self-cleavage C-terminal fusion system was developed using the inositol hexakisphosphate-inducible Vibrio cholerae MARTXVc toxin cysteine protease domain (CPD). CPD fusion proteins are expressed from the T7 promoter and purified using a 6xHis-tag with immobilized-metal affinity chromatography. The C-terminal CPD-tag is removed by self-cleavage at the final purification stage. Here, we describe an efficient cloning method using Gibson assembly, followed by expression and purification of tagless recombinant proteins of interest using CPD self-cleavage.

The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypical mechanism to disrupt Ras-ERK signaling.

The Ras-extracellular signal-regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor-mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target.

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

BACKGROUND: Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. RESULTS: In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. CONCLUSION: pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

SIRT2 deletion enhances KRAS-induced tumorigenesis in vivo by regulating K147 acetylation status.

The observation that cellular transformation depends on breaching a crucial KRAS activity threshold, along with the finding that only a small percentage of cellsharboring KRAS mutations are transformed, support the idea that additional, not fully uncovered, regulatory mechanisms may contribute to KRAS activation. Here we report that KrasG12D mice lacking Sirt2 show an aggressive tumorigenic phenotype as compared to KrasG12D mice. This phenotype includes increased proliferation, KRAS acetylation, and activation of RAS downstream signaling markers. Mechanistically, KRAS K147 is identified as a novel SIRT2-specific deacetylation target by mass spectrometry, whereas its acetylation status directly regulates KRAS activity, ultimately exerting an impact on cellular behavior as revealed by cell proliferation, colony formation, and tumor growth. Given the significance of KRAS activity as a driver in tumorigenesis, identification of K147 acetylation as a novel post-translational modification directed by SIRT2 in vivo may provide a better understanding of the mechanistic link regarding the crosstalk between non-genetic and genetic factors in KRAS driven tumors.

Site-specific processing of Ras and Rap1 Switch I by a MARTX toxin effector domain.

Ras (Rat sarcoma) protein is a central regulator of cell growth and proliferation. Mutations in the RAS gene are known to occur in human cancers and have been shown to contribute to carcinogenesis. In this study, we show that the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin-effector domain DUF5(Vv) from Vibrio vulnificus to be a site-specific endopeptidase that cleaves within the Switch 1 region of Ras and Rap1. DUF5(Vv) processing of Ras, which occurs both biochemically and in mammalian cell culture, inactivates ERK1/2, thereby inhibiting cell proliferation. The ability to cleave Ras and Rap1 is shared by DUF5(Vv) homologues found in other bacteria. In addition, DUF5(Vv )can cleave all Ras isoforms and KRas with mutations commonly implicated in malignancies. Therefore, we speculate that this new family of Ras/Rap1-specific endopeptidases (RRSPs) has potential to inactivate both wild-type and mutant Ras proteins expressed in malignancies.

Induced autoprocessing of the cytopathic Makes caterpillars floppy-like effector domain of the Vibrio vulnificus†MARTX toxin.

The multifunctional-autoprocessing repeats-in-toxin (MARTX(Vv)) toxin that harbours a varied repertoire of effector domains is the primary virulence factor of Vibrio vulnificus. Although ubiquitously present among Biotype I toxin variants, the 'Makes caterpillars floppy-like' effector domain (MCF(Vv)) is previously unstudied. Using transient expression and protein delivery, MCF(Vv) and MCF(Ah) from the Aeromonas hydrophila MARTX(Ah)) toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C-terminal subdomain of MCF(Vv) identified an Arg-Cys-Asp (RCD) tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCF(Vv). The autoprocessing could be recapitulated in vitro by the addition of host cell lysate to recombinant MCF(Vv), indicating induced autoprocessing by cellular factors. The RCD motif is also essential for cytopathicity, suggesting autoprocessing is essential first to activate the toxin and then to process a cellular target protein resulting in cell rounding. Sequence homology places MCF(Vv) within the C58 cysteine protease family that includes the type III secretion effectors YopT from Yersinia spp. and AvrPphB from Pseudomonas syringae. However, the catalytic site RCD motif is unique compared with other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins.

Role of ARF6, Rab11 and external Hsp90 in the trafficking and recycling of recombinant-soluble Neisseria meningitidis adhesin A (rNadA) in human epithelial cells.

Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.

Cytotoxicity of the Vibrio vulnificus MARTX toxin effector DUF5 is linked to the C2A subdomain.

The multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins are bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species' MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full-length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate-3721 and arginine-3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5(Ah) domain was also cytotoxic, while the weakly conserved C1-C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain.