Considering Protonation as a Posttranslational Modification Regulating Protein Structure and Function

Post-translational modification of proteins is an evolutionarily conserved mechanism for regulating activity, binding affinities and stability. Compared with established post-translational modifications such as phosphorylation or uniquitination, post-translational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, post-translational modification by protons can drive dynamical changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and in contrast to most other post-translational modifications does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton post-translational modification of pH-sensing proteins regulating different cellular processes

Binding of hisactophilin I and II to lipid membranes is controlled by a pH-dependent myristoyl-histidine switch

The interaction of the two N-terminally myristoylated isoforms of Dictyostelium hisactophilin with lipid model membranes was investigated by means of the monolayer expansion method and high-sensitivity titration calorimetry. The two isoforms, hisactophilin I and hisactophilin II, were found to insert with their N-terminal myristoyl residue into an electrically neutral POPC monolayer corresponding in its lateral packing density to that of a lipid bilayer. The partition coefficient for this insertion process was Kp = (1.1 +/- 0.2) x 10(4) M-1. The area requirement of the protein in the lipid membrane was estimated as 44 +/- 6 A2 which corresponds to the cross sectional area of the myristoyl moiety with an additional small contribution from amino acid side chains. The interaction of hisactophilin I (hisactophilin II) with negatively charged membrane surfaces is modulated in a pH-dependent manner by charged amino acid residues clustered around the myristoyl moiety. The electrostatic binding site consists of three lysine (one arginine and two lysine), seven (nine) histidine, and four (four) glutamic acid residues and has an isoelectric point of 6.9 (7.1). For small unilamellar POPC/POPG (75/25 mole/mole) vesicles, an apparent binding constant, K(app) = (8 +/- 1) x 10(5) M-1, was measured at pH 6.0 by means of high-sensitivity titration calorimetry. Electrostatic interactions hence increase the binding constant by about 2 orders of magnitude compared to hydrophobic binding alone. With increasing pH, the electrostatic attraction decreases and turns into an electrostatic repulsion at pH < 7.0 +/- 0.1. The area occupied by the cluster of charged residues constituting the membrane binding region was 280 +/- 20 A2 as derived from monolayer measurements in close agreement with molecular modeling data derived from the NMR structure of hisactophilin I [Habazettl et al. (1992) Nature 359, 855-858]

In vitro and in vivo activity of MT201, a fully human monoclonal antibody for pancarcinoma treatment

In our study, a novel, fully human, recombinant monoclonal antibody of the IgG1 isotype, called MT201, was characterized for its binding properties, complement-dependent (CDC) and antibody-dependent cellular cytotoxicity (ADCC), as well as for its in vivo antitumor activity in a nude mouse model. MT201 was found to bind its target, the epithelial cell adhesion molecule (Ep-CAM; also called 17-1A antigen, KSA, EGP-2, GA733-2), with low affinity in a range similar to that of the clinically validated, murine monoclonal IgG2a antibody edrecolomab (Panorex®). MT201 exhibited Ep-CAM-specific CDC with a potency similar to that of edrecolomab. However, the efficacy of ADCC of MT201, as mediated by human immune effector cells, was by 2 orders of magnitude higher than that of edrecolomab. Addition of human serum reduced the ADCC of MT201 while it essentially abolished ADCC of edrecolomab within the concentration range tested. In a nude mouse xenograft model, growth of tumors derived from the human colon carcinoma line HT-29 was significantly and comparably suppressed by MT201 and edrecolomab. The fully human nature and the improved ADCC of MT201 with human effector cells will make MT201 a promising candidate for the clinical development of a novel pan-carcinoma antibody that is superior to edrecolomab

Role of cortical tension in bleb growth

Blebs are spherical membrane protrusions often observed during cell migration, cell spreading, cytokinesis, and apoptosis, both in cultured cells and in vivo. Bleb expansion is thought to be driven by the contractile actomyosin cortex, which generates hydrostatic pressure in the cytoplasm and can thus drive herniations of the plasma membrane. However, the role of cortical tension in bleb formation has not been directly tested, and despite the importance of blebbing, little is known about the mechanisms of bleb growth. In order to explore the link between cortical tension and bleb expansion, we induced bleb formation on cells with different tensions. Blebs were nucleated in a controlled manner by laser ablation of the cortex, mimicking endogenous bleb nucleation. Cortical tension was modified by treatments affecting the level of myosin activity or proteins regulating actin turnover. We show that there is a critical tension below which blebs cannot expand. Above this threshold, the maximal size of a bleb strongly depends on tension, and this dependence can be fitted with a model of the cortex as an active elastic material. Together, our observations and model allow us to relate bleb shape parameters to the underlying cellular mechanics and provide insights as to how bleb formation can be biochemically regulated during cell motility