Tuft Cell Formation Reflects Epithelial Plasticity in Pancreatic Injury: Implications for Modeling Human Pancreatitis.

Chronic pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma (PDA), is a serious, widespread medical condition characterized by inflammation, fibrosis, and acinar to ductal metaplasia (ADM). ADM is a cell type transdifferentiation event where pancreatic acinar cells become ductal-like under conditions of injury or oncogenic mutation. Here, we show that chronic pancreatitis and ADM in genetically wild type mice results in the formation of a significant population of chemosensory tuft cells. Transcriptomic analyses of pancreatitis tuft cells identify expression of inflammatory mediators, consistent with a role for tuft cells in injury progression and/or resolution. Though similar to tuft cell populations in other organs and disease systems, we identified a number of key differences that suggest context-specific tuft cell functions. We evaluated seven different mouse strains for tuft cell formation in response to chronic injury and identified significant heterogeneity reflecting varying proclivity for epithelial plasticity between strains. These results have interesting implications in the role of epithelial plasticity and heterogeneity in pancreatitis and highlight the importance of mouse strain selection when modeling human disease.

Immunohistochemistry (IHC): Chromogenic Detection of 3-Phosphohistidine Proteins in Formaldehyde-Fixed, Frozen Mouse Liver Tissue Sections.

The development of antibodies that specifically detect histidine-phosphorylated proteins is a recent achievement and allows potential roles of histidine phosphorylated proteins in pathological and physiological conditions to be characterized. Immunohistochemical analyses enable the detection of proteins in tissues and can reveal alterations to the quantity and/or localization of these proteins through comparisons of normal and diseased specimens. However, the sensitivity of phosphohistidine modifications to phosphatases, acidic pH, and elevated temperatures poses unique challenges to the detection process and requires a protocol that bypasses traditional procedures utilizing paraffin-embedding and antigen-retrieval methods. Here, we detail a method for a brief fixation by 4% (v/v) paraformaldehyde on freshly collected tissues in the presence of PhosSTOP to block phosphatase activity, followed by a float on sucrose to protect the tissue prior to freezing. Specimens are then embedded in a cryopreservation medium in molds and frozen using an isoflurane, dry ice bath to best preserve the tissue morphology and phosphohistidine signal. We validate this technique in normal mouse liver using SC44-1, a monoclonal anti-3-pHis antibody used to uncover a role for a protein histidine phosphatase as a tumor suppressor in the liver. Furthermore, we demonstrate that the antibody signal can be eliminated by preincubating SC44-1 with a peptide treated with phosphoramidate to phosphorylate histidine residues. Thus, we present an IHC protocol suitable for specific detection of 3-phosphohistidine proteins in mouse liver tissue, and suggest that this can be used as a starting point for optimization of IHC using other phosphohistidine antibodies or in other tissue types, generating information that will enhance our understanding of phosphohistidine in models of disease.

Failure to detect functional transfer of active K-Ras protein from extracellular vesicles into recipient cells in culture.

Exosomes, extracellular nanovesicles that carry nucleic acids, lipids, and proteins, have been the subject of several studies to assess their ability to transfer functional cargoes to cells. We recently characterized extracellular nanovesicles released from glioblastoma cells that carry active Ras in complex with proteins regulating exosome biogenesis. Here, we investigated whether a functional transfer of Ras from exosomes to other cells can initiate intercellular signaling. We observed that treatment of serum-starved, cultured glioblastoma cells with exogenous glioblastoma exosomes caused a significant increase in cellular viability over time. Moreover, we detected fluorescent signal transfer from lipophilic dye-labeled exogenous glioblastoma exosomes into cultured glioblastoma cells. To probe possible signaling from cell-to-cell, we utilized bimolecular luciferase complementation to examine the ability of K-Ras in exosomes to interact with the Raf-Ras Binding domain (Raf-RBD) expressed in a recipient cell line. Although the K-Ras/Raf-RBD interaction was readily detectable upon co-expression in a single cell line, or following lysis of co-cultured cell lines separately expressing K-Ras and RBD, bearing in mind the limitations of our assay, we were unable to detect the interaction in the intact, co-cultured cell lines or upon treatment of the Raf-RBD-expressing cells with exosomes containing K-Ras. Furthermore, HA-Tag-BFP fused to the K-Ras hypervariable region and CAAX sequence failed to be transferred at significant levels from extracellular vesicles into recipient cells, but remained detectable in the cell supernatants even after 96 hours of culture of naïve cells with extracellular vesicles. We conclude that if transfer of functional K-Ras from extracellular vesicles into the cytoplasm of recipient cells occurs, it must do so at an extremely low efficiency and therefore is unlikely to initiate Ras-ERK MAP kinase pathway signaling. These results suggest that studies claiming functional transfer of protein cargoes from exosomes should be interpreted with caution.

Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner.

Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states.

Structure-function analysis of Rny1 in tRNA cleavage and growth inhibition.

T2 ribonucleases are conserved nucleases that affect a variety of processes in eukaryotic cells including the regulation of self-incompatibility by S-RNases in plants, modulation of host immune cell responses by viral and schistosome T2 enzymes, and neurological development and tumor progression in humans. These roles for RNaseT2's can be due to catalytic or catalytic-independent functions of the molecule. Despite this broad importance, the features of RNaseT2 proteins that modulate catalytic and catalytic-independent functions are poorly understood. Herein, we analyze the features of Rny1 in Saccharomyces cerevisiae to determine the requirements for cleaving tRNA in vivo and for inhibiting cellular growth in a catalytic-independent manner. We demonstrate that catalytic-independent inhibition of growth is a combinatorial property of the protein and is affected by a fungal-specific C-terminal extension, the conserved catalytic core, and the presence of a signal peptide. Catalytic functions of Rny1 are independent of the C-terminal extension, are affected by many mutations in the catalytic core, and also require a signal peptide. Biochemical flotation assays reveal that in rny1Δ cells, some tRNA molecules associate with membranes suggesting that cleavage of tRNAs by Rny1 can involve either tRNA association with, or uptake into, membrane compartments.

T2 Family ribonucleases: ancient enzymes with diverse roles.

Ribonucleases of the T2 family are found in the genomes of protozoans, plants, bacteria, animals and viruses. A broad range of biological roles for these ribonucleases have been suggested, including scavenging of nucleic acids, degradation of self-RNA, serving as extra- or intracellular cytotoxins, and modulating host immune responses. Recently, RNaseT2 family members have been implicated in human pathologies such as cancer and parasitic diseases. Interestingly, certain functions of RNaseT2 family members are independent of their nuclease activity, suggesting that these proteins have additional functions. Moreover, humans lacking RNASET2 manifest a defect in neurological development, perhaps due to aberrant control of the immune system. We review the basic structure and function of RNaseT2 family members and their biological roles.

LSM1 over-expression in Saccharomyces cerevisiae depletes U6 snRNA levels.

Lsm1 is a component of the Lsm1-7 complex involved in cytoplasmic mRNA degradation. Lsm1 is over-expressed in multiple tumor types, including over 80% of pancreatic tumors, and increased levels of Lsm1 protein have been shown to induce carcinogenic effects. Therefore, understanding the perturbations in cell process due to increased Lsm1 protein may help to identify possible therapeutics targeting tumors over-expressing Lsm1. Herein, we show that LSM1 over-expression in the yeast Saccharomyces cerevisiae inhibits growth primarily due to U6 snRNA depletion, thereby altering pre-mRNA splicing. The decrease in U6 snRNA levels causes yeast strains over-expressing Lsm1 to be hypersensitive to loss of other proteins required for production or function of the U6 snRNA, supporting a model wherein excess Lsm1 reduces the availability of the Lsm2-7 proteins, which also assemble with Lsm8 to form a complex that binds and stabilizes the U6 snRNA. Yeast strains over-expressing Lsm1 also display minor alterations in mRNA decay and demonstrate increased susceptibility to mutations inhibiting cytoplasmic deadenylation, a process required for both 5'-to-3' and 3'-to-5' pathways of exonucleolytic decay. These results suggest that inhibition of splicing and/or deadenylation may be effective therapies for Lsm1-over-expressing tumors.

Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes.

Ubiquitination directs the sorting of cell surface receptors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the removal of ubiquitin from MVB cargoes. Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination by Doa4 in the MVB pathway.