Activity and Synergy of Cu-ATCUN Antimicrobial Peptides.

Antibiotic resistance demands innovative strategies and therapies. The pairs of antimicrobial peptides tested in this work show broad-spectrum synergy and are capable of interacting with diverse bacterial membranes. In most cases, the ATCUN motif enhanced the activity of peptides tested in combination. Our studies also show CP10A to be a multifaceted peptide, displaying both cell membrane and intracellular activity and acting as a chameleon, improving the activity of other peptides as needed. The results of the synergy experiments demonstrate the importance of varied modes of action and how these changes can affect the ability to combat pathogens, while also illustrating the value of the metal-binding domain in enhancing the activity of antimicrobial peptides in combination.

Tackling antimicrobial stewardship through synergy and antimicrobial peptides.

The unrestricted use of antibiotics has led to rapid development of antibiotic resistance (AR) and renewed calls to address this serious problem. This review summarizes the most common mechanisms of antibiotic action, and in turn antibiotic resistance, as well as pathways to mitigate the harm. Focus is then turned to emerging antibiotic strategies, including antimicrobial peptides (AMPs), with a discussion of their modes of action, biochemical features, and potential challenges for their use as antibiotics. The role of synergy in antimicrobials is also examined, with a focus on the synergy of AMPs and other emerging interactions with synergistic potential.

Active site characterization and activity of the human aspartyl (asparaginyl) ß-hydroxylase.

Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the ß-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

Human aspartyl (asparaginyl) hydroxylase. A multifaceted enzyme with broad intra- and extra-cellular activity.

Human aspartyl (asparaginyl) ß-hydroxylase (HAAH), a unique iron and 2-oxoglutarate dependent oxygenase, has shown increased importance as a suspected oncogenic protein. HAAH and its associated mRNA are upregulated in a wide variety of cancer types, however, the current role of HAAH in the malignant transformation of cells is unknown. HAAH is suspected to play an important role in NOTCH signaling via selective hydroxylation of aspartic acid and asparagine residues of epidermal growth factor (EGF)-like domains. HAAH hydroxylation also potentially mediates calcium signaling and oxygen sensing. In this review, we summarize the current state of understanding of the biochemistry and chemical biology of this enzyme, identify key differences from other family members, outline its broader intra- and extra-cellular roles, and identify the most promising areas for future research efforts.

Enhanced Synergism and Mechanism of Action Studies of Synthetic Antimicrobial Metallopeptides.

Antimicrobial peptides (AMPs) are found throughout most kingdoms of life, are an important part of host immunity, and have been shown to act synergistically in various organisms to ameliorate bacterial infections. Herein, we report the synergistic behavior observed between two AMPs, Sub5 and CP10A, against E. coli. In addition, enhanced synergistic activity against E. coli and MRSA 43300 for two derivatives of Sub5, extended with the amino-terminal copper and nickel (ATCUN) binding motif, is observed when dosed together with CP10A, while displaying little cytotoxicity towards human dermal fibroblasts. All three combinations of peptides co-localized within bacterial cells as evidenced by fluorescence confocal microscopy. Investigations into the mechanism of synergy shows that all peptides indirectly damage DNA within cells, while only the ATCUN derivatives can oxidize phospholipids. Combinations of peptides were also shown to upregulate the concentration of reactive oxygen species within both E. coli and MRSA 43300. These results suggest that the production of reactive oxygen species is an important aspect mechanistically and further highlights the potential of these metallopeptides to aid in the treatment of antibiotic-resistant infections.