Developing proteomic biomarkers for bladder cancer: towards clinical application.

Clinical use of proteomic biomarkers has the potential to substantially improve the outcomes of patients with bladder cancer. An unmet clinical need evidently exists for noninvasive biomarkers, which might enable improvements in both the diagnosis and prognosis of patients with bladder cancer, as well as improved monitoring of patients for the presence of recurrence. Urine is considered the optimal noninvasive source of proteomic biomarkers in patients with bladder cancer. Currently, a number of single-protein biomarkers have been detected in urine and tissue using a variety of proteomic techniques, each having specific conceptual considerations and technical implications. Promising preclinical data are available for several of these proteins; however, the combination of single urinary proteins into multimarker panels might better encompass the molecular heterogeneity of bladder cancer within this patient population, and prove more effective in clinical use.

Radical S-adenosylmethionine enzyme catalyzed thioether bond formation in sactipeptide biosynthesis.

Sactipeptides represent a new emerging class of ribosomally assembled and posttranslationally modified peptides that show diverse bioactivities. Their common hallmark is an intramolecular thioether bond that crosslink the sulfur atom of a cysteine residue with the α-carbon of an acceptor amino acid. This review summarizes recent achievements concerning the biosynthesis of sactipeptides in general and with special focus on the common enzymatic radical SAM mechanism leading to the thioether linkage formation. In addition this mechanism is compared to the mechanism of thioether bond formation during lanthipeptide biosynthesis and to other radical based thioether bond forming reactions.

Two [4Fe-4S] clusters containing radical SAM enzyme SkfB catalyze thioether bond formation during the maturation of the sporulation killing factor.

The sporulation killing factor (SKF) is a 26-residue ribosomally assembled and posttranslationally modified sactipeptide. It is produced by Bacillus subtilis 168 and plays a key role in its sporulation. Like all sactipeptides, SKF contains a thioether bond, which links the cysteine residue Cys4 with the α-carbon of the methionine residue Met12. In this study we demonstrate that this bond is generated by the two [4Fe-4S] clusters containing radical SAM enzyme SkfB, which is encoded in the skf operon. By mutational analysis of both cluster-binding sites, we were able to postulate a mechanism for thioether generation which is in agreement with that of AlbA. Furthermore, we were able to show that thioether bond formation is specific toward hydrophobic amino acids at the acceptor site. Additionally we demonstrate that generation of the thioether linkage is leader-peptide-dependent, suggesting that this reaction is the first step in SKF maturation.

The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A.

Subtilosin A is a 35-residue, ribosomally synthesized bacteriocin encoded by the sbo-alb operon of Bacillus subtilis. It is composed of a head-to-tail circular peptide backbone that is additionally restrained by three unusual thioether bonds between three cysteines and the α-carbon of one threonine and two phenylalanines, respectively. In this study, we demonstrate that these bonds are synthesized by the radical S-adenosylmethionine enzyme AlbA, which is encoded by the sbo-alb operon and comprises two [4Fe-4S] clusters. One [4Fe-4S] cluster is coordinated by the prototypical CXXXCXXC motif and is responsible for the observed S-adenosylmethionine cleavage reaction, whereas the second [4Fe-4S] cluster is required for the generation of all three thioether linkages. On the basis of the obtained results, we propose a new radical mechanism for thioether bond formation. In addition, we show that AlbA-directed substrate transformation is leader-peptide dependent, suggesting that thioether bond formation is the first step during subtilosin A maturation.