Predicting sulfotyrosine sites using the random forest algorithm with significantly improved prediction accuracy

addresses: School of Biosciences, University of Exeter, Exeter EX4 5DE, UK. [email protected]: PMCID: PMC2777180types: Journal Article; Research Support, Non-U.S. Gov't© 2009 Yang; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Tyrosine sulfation is one of the most important posttranslational modifications. Due to its relevance to various disease developments, tyrosine sulfation has become the target for drug design. In order to facilitate efficient drug design, accurate prediction of sulfotyrosine sites is desirable. A predictor published seven years ago has been very successful with claimed prediction accuracy of 98%. However, it has a particularly low sensitivity when predicting sulfotyrosine sites in some newly sequenced proteins

A potential antibody repertoire diversification mechanism through tyrosine sulfation for biotherapeutics engineering and production

The diversity of three hypervariable loops in antibody heavy chain and light chain, termed the complementarity-determining regions (CDRs), defines antibody’s binding affinity and specificity owing to the direct contact between the CDRs and antigens. These CDR regions typically contain tyrosine (Tyr) residues that are known to engage in both nonpolar and pi stacking interaction with antigens through their complementary aromatic ring side chains. Nearly two decades ago, sulfotyrosine residue (sTyr), a negatively charged Tyr formed by Golgi-localized membrane-bound tyrosylprotein sulfotransferases during protein trafficking, were also found in the CDR regions and shown to play an important role in modulating antibody-antigen interaction. This breakthrough finding demonstrated that antibody repertoire could be further diversified through post-translational modifications, in addition to the conventional genetic recombination. This review article summarizes the current advances in the understanding of the Tyr-sulfation modification mechanism and its application in potentiating protein-protein interaction for antibody engineering and production. Challenges and opportunities are also discussed

In Silico Veritas: The Pitfalls and Challenges of Predicting

Recently the first community-wide assessments of the prediction of the structures of complexes between proteins and small molecule ligands have been reported in the so-called GPCR Dock 2008 and 2010 assessments. In the current review we discuss the different steps along the protein-ligand modeling workflow by critically analyzing the modeling strategies we used to predict the structures of protein-ligand complexes we submitted to the recent GPCR Dock 2010 challenge. These representative test cases, focusing on the pharmaceutically relevant G Protein-Coupled Receptors, are used to demonstrate the strengths and challenges of the different modeling methods. Our analysis indicates that the proper performance of the sequence alignment, introduction of structural adjustments guided by experimental data, and the usage of experimental data to identify protein-ligand interactions are critical steps in the protein-ligand modeling protocol. © 2011 by the authors; licensee MDPI, Basel, Switzerland

A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface

Retrograde transport of membranes and proteins from the cell surface is essential to maintain homeostasis and compartment identity. Following internalization via clathrin-dependent or -independent endocytosis, lipid and protein cargoes first populate early endosomes from where they are further redirected either along the endo-lysosomal system, recycled to the plasma membrane, or targeted to the trans-Golgi network (TGN) compartment. A number of distinct sorting machineries have been implicated in retrograde transport from endosomes to the TGN, among them the AP-1/clathrin machinery. Apart from an involvement in retrograde transport, AP-1/clathrin carriers have a well-established function in cargo export from the TGN. Even though the concept of bidirectional traffic at the TGN-to-endosome interface is commonly accepted, there is still uncertainty about the precise contribution of AP-1 to retrograde transport, since the conclusions of most studies were based on altered receptor steady-state distribution or mislocalization analysis upon knockdown or knockout of AP-1. Their readouts may be misleading, because the observed phenotype may be an indirect consequence of long-term AP-1 depletion, the result of upregulation of alternative pathways to compensate for the reduced or missing protein, thereby potentially masking the true AP-1 phenotype. To elucidate the involvement of AP-1 in endosome-to-TGN traffic, we set up a more generic approach allowing us to follow cargo molecules during their retrograde transport from the plasma membrane. To this end, we established a versatile nanobody-based approach conferring recombinant protein cargo to be tracked from the cell surface biochemically, by live cell imaging, and by electron microscopy. We engineered and bacterially expressed functionalized anti-GFP nanobodies fused to a sulfation consensus motif, to fluorophores, or to a peroxidase reporter. These functionalized nanobodies are specifically captured by EGFP-modified receptor proteins at the cell surface and transported piggyback to the receptor’s homing compartments. Using the sulfatable nanobody, we could biochemically determine the kinetics of bonafide sorting receptors, the MPRs, from the cell surface to the TGN. In combination with the knocksideways approach to look at the immediate and direct consequences of AP-1 inactivation, we could also show the role of AP-1/clathrin carriers in retrograde transport of MPRs from endosomes to the TGN. At the same time, however, we also evidenced that an AP-1 knockdown and knockout produced conflicting results when compared to acute inactivation strategies. Collectively, the present study describes a versatile nanobody-based approach to analyze retrograde transport of cargo proteins from the cell surface, and moreover provides insights into the role of the AP-1/clathrin machinery in retrograde transport

Structural and mechanistic studies of post-translationally modified peptides and proteins.

In mass spectrometry (MS), negative ions can be formed by many ion sources, and although sometimes less predominant than their cationic counterparts, they can be observed and studied to provide complementary molecular, ionic structure and mechanistic information. The research presented in this thesis investigates the production and use of negative ions for the structural determination of underivatised peptides and proteins and some post-translationally modified peptides and proteins. An additional application of this research is to determine the structure and membrane interaction of some peptides isolated from Australian amphibians. Phosphorylated Tyr (pTyr) containing peptides undergo SNi [N subscript] cyclisation of the C-terminal carboxylate anion at the P of the pTyr to effect transfer of PO₃H₂ to the C-terminal position. (A similar phosphate rearrangement from pTyr to side-chain carboxylate sites or to the side chains of Ser/Thr also occurs). Following proton transfer, several rearrangements initiated by this phosphate anion can occur, including a specific cyclisation to, and cleavage of, the peptide backbone at the central C of the penultimate amino acid residue. When a peptide contains two/three phosphate side chains, phosphate groups undergo phosphate/phosphate cyclisation to form characteristic di-/tri-phosphate anions. The mechanisms of all fragmentation processes are suggested with the assistance of ab initio theoretical calculations. The major negative-ion fragmentation of Tyr sulfate containing peptides is [(M-H) - SO₃]⁻ and this process normally yields the base peak of the spectrum. Rearrangement reactions involving the formation of HOSO₃⁻ and [(M-H) - H₂SO₄]⁻ yield minor peaks with relative abundances ≤ 10% and ≤ 2% respectively. A Ser sulfate containing peptide, in contrast, shows pronounced peaks due to cleavage product anions [(M-H) - SO₃]⁻ and HOSO₃⁻. Theoretical calculations at the CAM-B3LYP/6-311++g(d,p) level of theory suggest that rearrangement of a Ser sulfate to give C-terminal CO2SO3H is energetically unfavourable in comparison with fragmentation of the intact Ser sulfate to yield [(M-H) - SO₃]⁻ and HOSO₃⁻. [(M-H) - H₂SO₄]⁻ anions are not observed in the spectra of peptides containing Ser sulfate, presumably because HOSO₃⁻ is a relatively weak gas-phase base (∆Gacid = 1265 kJ mol⁻¹). The peaks corresponding to anions formed following cyclisation of the sulfate groups are not detected in the spectra of energised (M-H)⁻ ions of Ser disulfate containing peptides. Proteolytic digest/negative ion nanospray MS was used to determine the five disulfide units and much of the amino acid sequence of ricin, addressing both ricin detection and structural confirmation. Negative ion MS is found to be more effective than positive ion MS in identification and sequencing disulfide bridged peptides. While positive ion MS only provides partial sequences of disulfide containing peptides and often does not specify the positions of disulfide resides, negative ion MS gives clear evidence for the presence and positions of disulfide linkages via characteristic fragmentations. The skin peptide profiles of the red tree frog Litoria rubella (L. rubella) from three locations, namely Flinders Ranges, a region of south-western Queensland and Longreach (Queensland), have been investigated in an eight-month survey. Nine peptides were identified primarily using MS. While the secretion from the L. rubella frogs from Flinders Ranges consists of only the major peptide, tryptophyllin L1.2; the L. rubella frogs from the south-western Queensland and Longreach (Queensland) produce a number of small tryptophyllin peptides and two rubellidins (caeridin type). The primary structures of the major peptide tryptophyllin L1.2 and the two rubellidins (caeridin type) 4.1 and 4.2 were determined previously. The noticeable findings were the discovery of three tryptophyllin metabolite containing peptides including tryptophyllin L1.6, 1.7 and 1.8. The peptide profiles of these frog populations added more information about the evolutionary divergence of this genus. Schwyzer and Zerbe have proposed that certain neuropeptides can transfer from extracellular fluid to attach to a cell membrane prior to moving from that membrane to the adjacent active site of a transmembrane receptor. There are differences in the detailed mechanisms proposed but the key feature is the initial addition of the neuropeptide to the membrane. The Quartz Crystal Microbalance technique with Dissipation (QCM-D) was used to see whether certain amphibian neuropeptides are able to add to a mammalian model bilayer without destroying that membrane. It appears that the peptides may have different modes of interaction with the membrane depending upon overall charges, the charge densities, the secondary structures and the free energies of transferring (to water-membrane interface and to membrane interior), and that the membrane binding may take part but not play a requisite role in a receptor-binding process.Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 201