Tristan P. Wallis

Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways

Cells adapt to hypoxia by a cellular response, where hypoxia-inducible factor 1α (HIF-1α) becomes stabilized and directly activates transcription of downstream genes. In addition to this “canonical” response, certain aspects of the pathway require integration with Notch signaling, i.e., HIF-1α can interact with the Notch intracellular domain (ICD) to augment the Notch downstream response. In this work, we demonstrate an additional level of complexity in this cross-talk: factor-inhibiting HIF-1 (FIH-1) regulates not only HIF activity, but also the Notch signaling output and, in addition, plays a role in how Notch signaling modulates the hypoxic response. We show that FIH-1 hydroxylates Notch ICD at two residues (N1945 and N2012) that are critical for the function of Notch ICD as a transactivator within cells and during neurogenesis and myogenesis in vivo. FIH-1 negatively regulates Notch activity and accelerates myogenic differentiation. In its modulation of the hypoxic response, Notch ICD enhances recruitment of HIF-1α to its target promoters and derepresses HIF-1α function. Addition of FIH-1, which has a higher affinity for Notch ICD than for HIF-1α, abrogates the derepression, suggesting that Notch ICD sequesters FIH-1 away from HIF-1α. In conclusion, the data reveal posttranslational modification of the activated form of the Notch receptor and an intricate mode of cross-coupling between the Notch and hypoxia signaling pathways.

The Diversity of Bioactive Proteins in Australian Snake Venoms

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and α-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5′-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.

Molecular Diversity in Venom from the Australian Brown Snake, Pseudonaja textilis

Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A2s, and pre- and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two-dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, γ-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents.

Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% 18 O water

Determination of the disulfide‐bond arrangement of a protein by characterization of disulfide‐linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of 18O into peptides during peptic digestion of disulfide‐linked proteins in 50% 18O water resulted in isotope patterns and increases in average masses that facilitated ideication and characterization of disulfide‐linked peptides even in complex mixtures, without the need for reference digests in 100% 16O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix‐assisted laser desorption/ionization–time of flight (MALDI‐TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single‐chain peptides containing an intrachain disulfide bond, could be ideied and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide‐linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide‐bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.

Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate-interacting Proteins Using an Affinity Pulldown Strategy

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; α-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P2 binding, are important for chromaffin granule exocytosis.

Preparation and Analysis of Proteins and Peptides Using MALDI TOF/TOF Mass Spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry (MALDI‐TOF/TOF‐MS) is a valuable tool for the analysis of peptides and proteins. Particularly useful features include high sensitivity, fast data acquisition, ease of use, and robust instrumentation. Although MALDI is relatively tolerant to buffers and other impurities, substantial sensitivity enhancement can be achieved through removal of non‐analyte components of samples. Therefore, sample processing to remove buffers and impurities can greatly improve the quality of results obtained by MALDI experiments. This unit describes optimized procedures for enzymatic digestion, preparation of MALDI target plates, thin layer matrix preparation, on‐target sample cleanup, and capillary HPLC‐MALDI co‐spotting of analyte and matrix. Procedures are also described for analysis of on‐membrane proteins by MALDI‐TOF/TOF‐MS before tryptic digestion. Some of these procedures are also applicable to protein spots from two‐dimensional (2‐D) gels. Guidance is also provided for acquisition and interpretation of MS and MS/MS spectra. Curr. Protoc. Protein Sci. 63:16.13.1‐16.13.21.

Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp

Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp that is classified in the genus Okavirus, family Roniviridae, in the order Nidovirales. Separation of virion proteins treated with peptide-N-glycosidase-F (PNGase-F) in SDS-polyacrylamide gels and the use of glycoprotein-specific staining methods indicated that the gp116 and gp64 envelope glycoproteins possess N-linked rather than O-linked glycans. Competitive binding inhibition of lectins with various oligosaccharide specificities indicated that glycans linked to gp64 are mannose-rich, whilst glycans linked to gp116 possess terminal N-acetylgalactosamine and N-acetylglucosamine in addition to terminal mannose-type sugars. Mass spectrometry analyses of peptides generated from YHV proteins before and after deglycosylation with PNGase-F, using combinations of the endoproteinases trypsin, Asp-N and Lys-C, confirmed occupancy of six of the seven potential N-linked glycosylation sites in gp116 and three of the four potential sites in gp64.