We have previously reported that, within the first helix of human interleukin (IL)-3, residues Asp21 and Glu22 are important for interaction with the α- and β-chains of the IL-3 receptor, respectively. In order to define more precisely the sites of interaction with the receptor, we have performed molecular modeling of the helical core of IL-3 and single amino acid substitution mutagenesis of residues predicted to lie on the surfaces of the A, C, and D helices. The resulting analogues were characterized for their abilities to stimulate proliferation of TF-l cells and for binding to the high affinity (α- and β-chain; IL-3Rα/Rβ) or low affinity (α-chain alone; IL-3Rα) IL-3 receptor. We found that in addition to Asp21, residues Ser17, Asn18, and Thr25 within the A helix and Arg108, Phe113, Lys116, and Glu119 within the D helix of IL-3 were important for biological activity. Analysis of their binding characteristics revealed that these analogues were deficient in binding to both the IL-3Rα/Rβ and the IL-3Rα forms of the receptor, consistent with a selective impairment of interaction with IL-3Rα. Molecular modeling suggests that these eight amino acid residues are adjacent in the tertiary structure, consistent with a discontinuous epitope interacting selectively with IL-3Rα. On the other hand, Glu22 of IL-3 was found to interact preferentially with the β-chain with bulky and positively charged substitutions causing greater than 10,000-fold reduction in biological activity. These results show fundamental differences between IL-3 and granulocyte-macrophage colony-stimulating factor in the structural basis for recognition of their receptors that has implications for the construction of novel analogues and our understanding of receptor activation. We have previously reported that, within the first helix of human interleukin (IL)-3, residues Asp21 and Glu22 are important for interaction with the α- and β-chains of the IL-3 receptor, respectively. In order to define more precisely the sites of interaction with the receptor, we have performed molecular modeling of the helical core of IL-3 and single amino acid substitution mutagenesis of residues predicted to lie on the surfaces of the A, C, and D helices. The resulting analogues were characterized for their abilities to stimulate proliferation of TF-l cells and for binding to the high affinity (α- and β-chain; IL-3Rα/Rβ) or low affinity (α-chain alone; IL-3Rα) IL-3 receptor. We found that in addition to Asp21, residues Ser17, Asn18, and Thr25 within the A helix and Arg108, Phe113, Lys116, and Glu119 within the D helix of IL-3 were important for biological activity. Analysis of their binding characteristics revealed that these analogues were deficient in binding to both the IL-3Rα/Rβ and the IL-3Rα forms of the receptor, consistent with a selective impairment of interaction with IL-3Rα. Molecular modeling suggests that these eight amino acid residues are adjacent in the tertiary structure, consistent with a discontinuous epitope interacting selectively with IL-3Rα. On the other hand, Glu22 of IL-3 was found to interact preferentially with the β-chain with bulky and positively charged substitutions causing greater than 10,000-fold reduction in biological activity. These results show fundamental differences between IL-3 and granulocyte-macrophage colony-stimulating factor in the structural basis for recognition of their receptors that has implications for the construction of novel analogues and our understanding of receptor activation.
Proteomic matrix-assisted laser desorption/ionisation (MALDI) linear time-of-flight (TOF) mass spectrometry (MS) may be used to produce protein profiles from biological samples with the aim of discovering biomarkers for disease. However, the raw protein profiles suffer from several sources of bias or systematic variation which need to be removed via pre-processing before meaningful downstream analysis of the data can be undertaken. Baseline subtraction, an early pre-processing step that removes the non-peptide signal from the spectra, is complicated by the following: (i) each spectrum has, on average, wider peaks for peptides with higher mass-to-charge ratios (m/z), and (ii) the time-consuming and error-prone trial-and-error process for optimising the baseline subtraction input arguments. With reference to the aforementioned complications, we present an automated pipeline that includes (i) a novel `continuous' line segment algorithm that efficiently operates over data with a transformed m/z-axis to remove the relationship between peptide mass and peak width, and (ii) an input-free algorithm to estimate peak widths on the transformed m/z scale. The automated baseline subtraction method was deployed on six publicly available proteomic MS datasets using six different m/z-axis transformations. Optimality of the automated baseline subtraction pipeline was assessed quantitatively using the mean absolute scaled error (MASE) when compared to a gold-standard baseline subtracted signal. Near-optimal baseline subtraction was achieved using the automated pipeline. The advantages of the proposed pipeline include informed and data specific input arguments for baseline subtraction methods, the avoidance of time-intensive and subjective piecewise baseline subtraction, and the ability to automate baseline subtraction completely. Moreover, individual steps can be adopted as stand-alone routines.
Abstract Background HIV-1 reverse transcriptase (RT) is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome into DNA. RT can be post-translationally modified in vitro which may be an important mechanism for regulating RT activity. Here we report detection of different p66 and p51 RT isoforms by 2D gel electrophoresis in virions and infected cells. Results Major isoforms of the p66 and p51 RT subunits were observed, with pI's of 8.44 and 8.31 respectively (p66 8.44 and p51 8.31 ). The same major isoforms were present in virions, virus-infected cell lysates and intracellular reverse transcription complexes (RTCs), and their presence in RTCs suggested that these are likely to be the forms that function in reverse transcription. Several minor RT isoforms were also observed. The observed pIs of the RT isoforms differed from the pI of theoretical unmodified RT (p66 8.53 and p51 8.60 ), suggesting that most of the RT protein in virions and cells is post-translationally modified. The modifications of p66 8.44 and p51 8.31 differed from each other indicating selective modification of the different RT subunits. The susceptibility of RT isoforms to phosphatase treatment suggested that some of these modifications were due to phosphorylation. Dephosphorylation, however, had no effect on in vitro RT activity associated with virions, infected cells or RTCs suggesting that the phospho-isoforms do not make a major contribution to RT activity in an in vitro assay. Conclusion The same major isoform of p66 and p51 RT is found in virions, infected cells and RTC's and both of these subunits are post-translationally modified. This post-translational modification of RT may be important for the function of RT inside the cell.
The importance of Mn(2+) for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn(2+) is required are yet to be fully elucidated. Here, we analyzed the effect of Mn(2+) limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn(2+) transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn(2+)-dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn(2+)-SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn(2+) stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn(2+)-replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn(2+) having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn(2+) transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn(2+) stress.
The high affinity receptors for GM-CSF, IL-3 and IL-5 are heterodimers consisting of a ligand-specific alpha chain and a common beta chain. These proteins are members of a family of proteins known as the "cytokine receptor family" which is characterized by the presence of a 200-residue ligand-binding module. The GM-CSF, IL-3 and IL-5 receptor alpha chains constitute a distinct subgroup and share features not found in other members of the cytokine receptor family, features which we propose to be important for their interaction with the common beta chain and for their binding of the structurally-related ligands. The growth hormone receptor is a well-characterized member of the cytokine receptor family. Based on the structure of the complex between growth hormone and its receptor, we have proposed sites of contact between the GM-CSF, IL-3 and IL-5 receptors and their cognate ligands.
Abstract Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL-3) are cytokines active in both normal and abnormal hemopoiesis, inflammation, and immunity. Their biological activity is mediated via receptors that comprise a ligand-specific α chain and a β chain that is common to the GM-CSF, IL-3, and IL-5 receptors. To understand the mechanism of action of GM-CSF and IL-3 in both normal and pathological conditions, we are seeking to define the structural elements required for ligand/receptor and receptor/receptor contact and their role in cellular activation. To this end we have identified a conserved motif in the first helix of GM-CSF, Glu21 that is critical for high affinity binding and biological activity. Charge-reversal mutagenesis of this residue generates a GM-CSF analogue that is devoid of biological activity and can antagonize the activity of wild-type GM-CSF. This probably results from the selective deficiency in interaction with the β chain of the receptor and suggests that similar antagonists for IL-3 and IL-5 are also feasible. Complementary mutagenesis studies on the receptor β chain have identified the putative B’-C’ loop in the membrane-proximal domain as being critical for the high affinity binding of GM-CSF but not IL-3. Characterization of the specificity of sites of interaction between the ligands and receptors may permit the design of specific or generic antagonists that may have important therapeutic implications. J. Leukoc. Biol. 57: 739–746; 1995.
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