Posttranslational modification of proteins with ubiquitin and ubiquitin-like modifiers such as SUMO can be reverted by specific proteases, also referred to as deubiquitinases and isopeptidases, most of which are cysteine-dependent. We have found that the replacement of the conserved C-terminal glycine with propargylamine converts SUMO and ubiquitin to highly efficient covalent inhibitors of their cognate cysteine proteases. Attack of the catalytic cysteine onto the terminal alkyne results in the formation of a vinyl sulfide linkage. Although this reaction is reminiscent of the inhibitory mechanism of the isosteric nitrile inhibitors it was unexpected due to the low electrophilicity of the alkyne group. We show that a precise location of the functional group in the active site of the protease is crucial for the reaction, which was not inhibited by the presence of a radical scavenger. Furthermore, a mutational study of key catalytic residues in the SUMO-protease Senp1, that is H533A and D550A of the catalytic triad and Q597A as part of the oxyanion hole, revealed that these residues are not required for the observed covalent adduct formation. We therefore propose that the reaction is an in situ thiol-alkyne addition. Due to the high chemical inertness of the alkyne moiety the respective protease inhibitors should be well-suited for cellular and therapeutic applications. In keeping with this idea, selective labeling with propargylated SUMO and Ub probes was observed in lysates of cell lines expressing the cognate proteases after transient transfection.

Recent studies have shown that surfactant components, in particular the collectins surfactant protein (SP)-A and -D, modulate the phagocytosis of various pathogens by alveolar macrophages. This interaction might be important not only for the elimination of pathogens but also for the elimination of inhaled allergens and might explain anti-inflammatory effects of SP-A and SP-D in allergic airway inflammation. We investigated the effect of surfactant components on the phagocytosis of allergen-containing pollen starch granules (PSG) by alveolar macrophages. PSG were isolated from Dactylis glomerata or Phleum pratense, two common grass pollen allergens, and incubated with either rat or human alveolar macrophages in the presence of recombinant human SP-A, SP-A purified from patients suffering from alveolar proteinosis, a recombinant fragment of human SP-D, dodecameric recombinant rat SP-D, or the commercially available surfactant preparations Curosurf and Alveofact. Dodecameric rat recombinant SP-D enhanced binding and phagocytosis of the PSG by alveolar macrophages, whereas the recombinant fragment of human SP-D, SP-A, or the surfactant lipid preparations had no effect. In addition, recombinant rat SP-D bound to the surface of the PSG and induced aggregation. Binding, aggregation, and enhancement of phagocytosis by recombinant rat SP-D was completely blocked by EDTA and inhibited by d-maltose and to a lesser extent by d-galactose, indicating the involvement of the carbohydrate recognition domain of SP-D in these functions. The modulation of allergen phagocytosis by SP-D might play an important role in allergen clearance from the lung and thereby modulate the allergic inflammation of asthma.

Wrestling with SUMO: the chemical conjugation of proteins with small ubiquitin-like modifiers (SUMO) can be achieved by a copper(I)-catalyzed cycloaddition and unnatural amino acid mutagenesis. This approach overcomes previous restrictions related to the primary sequence of proteins and coupling conditions. Moreover, biochemical data suggests that this triazole linkage presents the modifier in a proper distance and orientation relative to the target protein.

A new route to the synthesis of triazole-linked ubiquitin dimers (diUbs) as structural analogs of the seven diUbs is reported. Binding studies with the Lys48-specific UBA domain of the Mud1 protein suggest that they represent functionally suitable surrogates of their native counterparts linked by an isopeptide bond.

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