Protamine as an efficient membrane-translocating peptide

Protamine, a mixture of positively charged proteins from salmon roe used in diverse pharmaceutical applications, was reacted with the N-hydroxysuccinimide ester of tetramethylrhodamine to yield tetramethylrhodamine-labeled protamines (Pro(Rh)) containing one mole of fluorochrome per mole of protein. The internalization of tetramethylrhodamine-labeled protamine (Pro(Rh)) and the fluorescein-labeled tat peptide (Tat(Fl)) showed a similar dependence on time and concentration. Pro(Rh) and Tat(Fl) showed strong nuclear localizations, evident with both live cells and fixed cells co-stained with DAPI, a nuclear stain. The loss of fluorescence when cells were loaded with Pro(Rh) or Tat(Fl) was similar, further supporting a strong similarity between these two materials. Finally, when Pro(Ph) was covalently attached to the amino-CLIO nanoparticle, the cellular uptake of the nanoparticle was greatly enhanced. All experiments were performed with HeLa and CaCo-2 cells with similar results. These observations imply that protamine, a protein in regular clinical use, might be used for the design of novel membrane translocating/nuclear localizing pharmaceuticals whose development was initiated with other membrane-translocating peptides. In addition, the fluorescent protamines developed here might be used to further our understanding of this important pharmaceutical.     

Induction of antitumor immunity by dendritic cells loaded with membrane-translocating mucin 1 Peptide antigen

To investigate the role of enhanced antigen presentation in dendritic cell (DC)-based immunotherapy. Here, we describe the development of a cell-penetrating mucin 1 (MUC1) antigen and its immunotherapeutic potential against tumors. After animal groups received two immunizations of MUC1-MPA(11)P-pulsed DCs, we observed a marked tumor regression compared with the mice treated with DCs alone or DCs pulsed with MUC1 peptide. We confirmed the migration and homing of DCs in the popliteal lymph node using magnetic resonance imaging during the study. In summary, enhanced antigen uptake using an MPA(11)P delivery molecule improves cell therapy.     

Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a β-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (α/α)₆ fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.     

Structural Snapshots of 26S Proteasome Reveal Tetraubiquitin-Induced Conformations

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Crystallographic Snapshots along the Reaction Pathway of Nucleoside Triphosphate Diphosphohydrolases

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Snapshots of a dynamic folding nucleus in zinc-substituted Pseudomonas aeruginosa azurin

Zinc-substituted Pseudomonas aeruginosa azurin folds in two-state equilibrium and kinetic reactions. In the unfolded state, the zinc ion remains bound to the unfolded polypeptide via two native-state ligands (His117 and Cys112). The significantly curved Chevron plot for zinc-substituted azurin was earlier ascribed to movement of the folding-transition state. At low concentrations of denaturant, the transition state occurs early in the folding reaction (low Tanford beta-value), whereas at high-denaturant concentration, it moves closer to the native structure (high Tanford beta-value). Here, we use this movement to track the formation and growth of zinc-substituted azurin's folding nucleus with atomic resolution using protein engineering. The average phi (phi) value for 17 positions (covering all secondary-structure elements) goes from 0.25 in 0 M GuHCl (beta approximately 0.46) to 0.76 in 4 M GuHCl (beta approximately 0.86); a phi-value of 1 or 0 indicates native-like or unfolded-like interactions, respectively. Analysis of individual phi-values reveals a delocalized nucleus where structure condenses around a leading density centered on Leu50 in the core. The diffuse moving transition state for zinc-substituted azurin is in sharp contrast to the fixed polarized folding nucleus observed for apo-azurin. The dramatic difference in apparent kinetic behavior for the two forms of azurin can be rationalized as a minor alteration on a common free-energy profile that exhibits a broad activation barrier.