TAP score: torsion angle propensity normalization applied to local protein structure evaluation

Background  

Experimentally determined protein structures may contain errors and require validation. Conformational criteria based on the Ramachandran plot are mainly used to distinguish bet ween distorted and adequately refined models. While the readily available criteria are sufficient to detect totally wrong structures, establishing the more subtle differences between plausible structures remains more challenging.     

The thermal tolerance of crown-of-thorns (Acanthaster planci) embryos and bipinnaria larvae: implications for spatial and temporal variation in adult populations

To understand the role of sea temperature on the population biology of the crown-of-thorns sea star Acanthaster planci, the thermal window for embryonic and larval development was investigated. In two experiments, the response of embryos and larvae across 12 temperatures from 19.4 to 36.5 °C was quantified as the percentage of individuals reaching cleavage stage embryos, blastula, gastrula, early-bipinnaria, late-bipinnaria larvae or abnormal. Measurements were made at 7 times up to 72 h post-fertilisation, with the morphometrics of larvae measured in the 72-h sample. Acanthaster planci developed at temperatures between 19.4 and 33.2 °C, with a thermal window for development to the late-bipinnaria stage between 25.6 and 31.6 °C. Development rate, normal development and larval size were optimal at 28.7 °C, with development rates remaining relatively constant up to 31.6 °C. Rates of abnormality increased steadily (early embryonic stages) above 28.7 °C and was 100 % at temperatures approaching 33 °C. These experiments provide a more detailed insight into the response of A. planci developmental stages to temperature. The present day distribution of the species in eastern Australia overlap with the optimal thermal window for development to the late-bipinnaria stage (≈25–32 °C), implying a role of temperature in controlling population distributions and abundances. Despite this, short- or long-term temperature increases may not be a major modulator of the crown-of-thorns recruitment success, population dynamics and distribution in the future as no significant change in development rates, larval survival and growth occurred within this thermal window. Therefore, moderate (1–2 °C) increases in sea temperatures caused by El Niño or near-future ocean warming may not drive an increase in developmental and settlement success. Indeed, without any acclimation to warmer temperatures expected under near-future warming (+2 to 4 °C), climate change could ultimately reduce larval survival due to elevated mortality above the optimal development temperature.     

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.     

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO₂H] and sulfonic [Cys-SO₃H] acids) that are considered “irreversible.” We developed an enrichment method to isolate Cys-SO₂H/SO₃H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pK_a Cys-SO₃H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO₂H/SO₃H-containing peptides from cationic resins (i.e. “negative” selection) followed by “positive” selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO₂H/SO₃H (up to 80%) from other modified peptides. We identified 181 Cys-SO₂H/SO₃H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H₂O₂ (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO₂H/SO₃H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair.Cysteine (Cys)¹ is an integral site for protein post-translational modification (PTM) in response to physiological and pathological stimuli. Numerous studies have identified roles for biologically reversible Cys PTM, including disulfides, S-nitrosothiols, and sulfenic acids (Cys-SOH), in the regulation of protein function during redox signaling (reviewed in (1, 2)). Additionally, Cys can be oxidized in pathologies associated with oxidative stress (e.g. neurodegeneration, cancer, and cardiovascular disease (2)). Various redox proteomics methods exist for enrichment of these reversibly oxidized Cys, based on reduction to the thiol and then capture by: 1) alkylation with a chemical tag (e.g. isotope coded affinity tags) (3–6); 2) thiol-disulfide exchange (7–10); or 3) heavy metal ion chelation (11, 12). Oxidative Cys PTMs with predominantly no known means of enzymatic reduction have also been identified. These “over” or “irreversibly” oxidized Cys PTM (sulfinic [Cys-SO₂H] and sulfonic [Cys-SO₃H] acids) are primarily associated with oxidative stress. Only one example of reversible Cys-SO₂H modification has been characterized—in peroxiredoxins (Prx) by the ATP-dependent sulfiredoxin (Srx)(13); however, Srx is not thought to reduce Cys-SO₂H in other proteins, and no mechanism has yet been found for Cys-SO₃H reduction. At basal levels, ∼1–2% of Cys exist as Cys-SO₂H/SO₃H (14), and the RSO₂H modification has functional significance in some proteins (e.g. DJ-1 is activated in Alzheimer''s disease by Cys-SO₂H at Cys-106) (15).Cys-SO₂H/SO₃H are produced via sequential oxidation of Cys-SOH, which itself is formed because of Cys thiol oxidation by reactive oxygen and nitrogen species (ROS/RNS), such as hydrogen peroxide (H₂O₂) or peroxynitrite. This reaction is relatively inefficient and requires three equivalents of oxidant, as well as the protection of the initial Cys-SOH from nucleophilic attack. Therefore, Cys forming these PTM, particularly at biologically relevant concentrations of oxidant, are likely to be highly reactive or located in a unique microenvironment that accommodates their production without prior reduction of the Cys-SOH (e.g. by thiol or amine attack). Such sites may thus be candidates as redox or regulatory sensors (reviewed in (16)). Alternatively, over-oxidation to Cys-SO₂H/SO₃H during elevated oxidative stress may serve as a marker of oxidative damage, and target proteins for degradation.Information on Cys-SO₂H/SO₃H PTM in complex samples has thus far been generated only by amino acid analysis (hydrolyzed lysates) (14) or two-dimensional gel electrophoresis (2-DE), where these PTM cause an acidic shift (17, 18). The former provides no information on specific proteins, whereas the latter relies on the modified population being of sufficient intensity for observation and/or the availability of antibodies against a protein-of-interest. A recent study identified 44 Cys-SO₂H/SO₃H-modified peptides in nonphysiologically H₂O₂ oxidized (440 μm) cells utilizing long column ultra-high pressure liquid chromatography (LC) (19). Global analysis of irreversible Cys-PTM thus requires enrichment that considers: (1) Cys is the second least abundant amino acid in proteins (∼1.5%) (20), and (2) Cys-SO₂H/SO₃H are expected to occupy only 1–2% of these Cys sites, under physiological (and perhaps even pathological) conditions.Specific peptide enrichment by LC followed by bottom-up proteomics is a common approach used successfully for many PTMs (21, 22). Limited studies, however, have explored such techniques for Cys-SO₂H/SO₃H-containing peptides, and none have examined complex lysates—only single purified proteins (23, 24). Given that these PTM are among the most acidic modifications, with an average pK_a of RSO₂H < 2 and RSO₃H ∼−3, it is pertinent to isolate these peptides by exploiting their unique charge distribution. At acidic pH, where nonmodified tryptic peptides will have an average in-solution charge state between one and two (depending on pK_a of acidic residues and the C terminus), Cys-SO₂H/SO₃H-containing peptides will have an added negative charge, and, thus, have average charge distribution ≤ 1. Selection can therefore be performed on either positively or negatively charged resins with the former being a “positive” selection for Cys-SO₂H/SO₃H-containing peptides (retained by the resin), whereas the latter is a “negative” selection (Cys-SO₂H/SO₃H-containing peptides will not be retained by the resin). Both approaches have been used (23, 24) to capture peptides from bovine serum albumin (BSA) oxidized by performic acid – causing scission of disulfide bonds and conversion of Cys to Cys-SO₃H, and methionine (Met) to the sulfone Met(O₂). The studies gave comparative results, with positive selection (24) increasing Cys coverage in comparison to negative selection (23) (60% versus 45%) at the expense of specificity, with more non-Cys peptides observed in the elution.Ultimately, any enrichment approach must be able to purify Cys-SO₂H/SO₃H-containing peptides from cells and tissues under physiological and/or pathological conditions, both of which will generate considerably lower levels of Cys-SO₂H/SO₃H than performic acid. Myocardial ischemia and reperfusion (I/R) injury is characterized by a “burst” of ROS/RNS that is observed upon reperfusion (25, 26). These ROS/RNS overwhelm the natural antioxidant defenses of the heart (27) and lead to oxidative stress that contributes to contractile dysfunction (28–30). Several studies have observed an increase in reversible Cys PTM following I/R (31–36), and an increase in Cys-SO₂H/SO₃H may also contribute to cellular dysfunction that ultimately leads to apoptosis and necrosis that follows prolonged I/R (myocardial infarction). Given the common practice of peptide fractionation with strong cation exchange (SCX) as a first dimension during bottom-up proteomics, we wished to explore its utility in identifying Cys-SO₂H/SO₃H sites in complex samples. Performic oxidized BSA and myocardial protein extracts were utilized to study the interactions occurring at each step of the method, and then the method was applied to myocardial protein extract that had been exposed to a high concentration of a less efficient oxidant (H₂O₂). Finally, the method was used to identify Cys-SO₂H/SO₃H-containing peptides derived from either physiologically relevant concentrations of H₂O₂ (i.e. ≤100 μm, an estimate of the likely pathological H₂O₂ levels (37, 38)) or from rat myocardial tissue subjected to I/R injury.     

The taste of polycose in hamsters

Hamsters show a preference for Polycose, a mixture of starch-derived glucose polymers, that is as strong as their preference for sucrose. However, in the hamster, taste aversions to Polycose may be less easily acquired than taste aversions to sucrose and the qualitative aspects of Polycose are unknown in this species. In order to examine the taste of Polycose in the hamster, we utilized a taste-aversion protocol with two conditioning trials. Animals were trained to avoid one of three different conditioning stimuli: 50 mM sucrose, 100 mM Polycose and a mixture of 50 mM sucrose with 100 mM Polycose. Control animals were conditioned with deionized water. After the second conditioning trial, generalization testing began for the three conditioning stimuli plus 3 mM citric acid, 300 mM KCI and 30 mM NaCl. The results showed that aversions to Polycose, sucrose or the Polycose/sucrose mixture cross- generalized, demonstrating that Polycose and sucrose share a common taste percept in the hamster. None of the aversions generalized to NaCl, citric acid or KCI. In addition, comparisons among the patterns of taste generalizations indicated that the tastes of Polycose and sucrose also had distinct qualitative components. Finally, although the taste of 100 mM Polycose was more salient than the taste of 50 mM sucrose, the taste of sucrose could still be detected in a mixture with Polycose.      

Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance

Galectins, beta-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of approximately 29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development.      

Epigenetic influences on genetically triggered thoracic aortic aneurysm

Genetically triggered thoracic aortic aneurysms (TAAs) account for 30% of all TAAs and can result in early morbidity and mortality in affected individuals. Epigenetic factors are now recognised to influence the phenotype of many genetically triggered conditions and have become an area of interest because of the potential for therapeutic manipulation. Major epigenetic modulators include DNA methylation, histone modification and non-coding RNA. This review examines epigenetic modulators that have been significantly associated with genetically triggered TAAs and their potential utility for translation to clinical practice.     

Monoclonal TCR-redirected tumor cell killing

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.