Protein scavenging by macropinocytosis can serve as a source of nutrients for pancreatic cancer cells. We provide direct evidence that extracellular protein is a fuel source for pancreatic cancer cells in vivo. We demonstrate that albumin-derived peptides and amino acids accumulate in tumors in a Kras-driven mouse model of pancreatic ductal adenocarcinoma. In addition, we implement a device to deliver large molecules directly into the tumor and observe protein catabolism and macropinocytosis by cancer cells within pancreatic tumors. Local release of a macropinocytosis inhibitor leads to a drastic reduction in amino acids levels in tumor tissue arguing that the direct uptake and catabolism of extracellular protein is necessary to provide amino acids to pancreatic cancer cells in tumors. These data provide evidence for albumin catabolism by tumors and also suggest a method for testing therapies that take advantage of the propensity of pancreatic cancer cells to scavenge extracellular protein.
HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 3 10 helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.antigen presentation | antigen processing | major histocompatibility proteins | chaperone | protein folding H LA-DM (DM) facilitates peptide exchange on class II MHC (MHC II) proteins, and is required for efficient peptide loading in vivo (1). MHC II molecules assemble in the endoplasmic reticulum with the class II-associated invariant chain chaperone, which is subsequently cleaved by endosomal proteases leaving a short fragment known as CLIP bound in the MHC peptidebinding groove (2). DM facilitates the exchange of CLIP for peptides generated by digestion of endogenous and exogenous proteins, resulting in a library of peptide antigens bound to MHC II proteins that are transported to the cell surface (3, 4). In vitro experiments have corroborated the roles for DM as a peptideexchange factor (5, 6) and as a molecular chaperone that prevents peptide-free MHC II molecules from becoming inactive and from forming aggregates (7,8).The mechanism by which DM mediates these effects has received much attention (7-17). Peptides are released by DM with different rates (18), with DM susceptibility a major factor in whether or not a particular peptide is recognized by the cellular immune system (19-21). Understanding the mechanism of DMmediated peptide release would promote efforts to predict immunogenicity of known and emerging pathogens. Moreover, the mechanism involves catalysis of a protein conformational change (16), and its elucidation would have implications for our understanding of protein-folding processes. However, despite intensive investigation and crystal structures of both MHC II and DM proteins, the mechanism of DM-facilitated MHC-peptide binding and exchange is not understood. Part of the difficulty in developing an understanding of the interaction of DM and MHC II may be because of the presence of multiple conformers of DM (22), as well as MHC II (10, 23). Previous studies using directed screens (11,14) and tethering approaches (24, 25) have identified residues from both DM and MHC II that are critical for the functional interaction. These residues include a cluster of acidic residues on DM (14) and several residues in the vicinity of the P1 pocket of MHC II (9,11,15). Models for the DM-MHC II complex that pla...
Drug transit through the blood-brain barrier (BBB) is essential for therapeutic responses in malignant glioma. Conventional methods for assessment of BBB penetrance require synthesis of isotopically labeled drug derivatives. Here, we report a new methodology using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) to visualize drug penetration in brain tissue without molecular labeling. In studies summarized here, we first validate heme as a simple and robust MALDI MSI marker for the lumen of blood vessels in the brain. We go on to provide three examples of how MALDI MSI can provide chemical and biological insights into BBB penetrance and metabolism of small molecule signal transduction inhibitors in the brain – insights that would be difficult or impossible to extract by use of radiolabeled compounds.
A top-down approach based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and beta-ethoxy-alpha-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chemistry to produce strand cleavage at the modified nucleotides, and analysis by polyacrylamide gel electrophoresis to determine the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the analysis of individual target adducts present in complex reaction mixtures with no need for separation techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MS(n)() experiments to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated reduction and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data reduction module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calculates the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data reduction program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on cross-linking strategies and top-down ESI-FTMS.
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