Exploring Cysteine-rich Botanical Bioactive Peptides via Mass Spectrometry

A rise in clinical multidrug-resistant microbes threatens global health, agriculture, and economy. Botanical natural product peptides are an underexplored source of antimicrobial therapeutics with novel and unique chemistries to infuse an increasingly dry drug pipeline. However, complex natural product matrices, inherent sequence variability, low abundance, and dynamic / circadian expression challenge the identification of novel antimicrobial peptides (AMPs): analytical methods are needed to explore the expansive chemical space offered by natural product AMPs. Mass spectrometry (MS) drives primary natural product discovery, where modern instruments coupled with ultra-high performance liquid chromatography (UHPLC) can identify and resolve low abundance peptidyl species in complex mixtures with unprecedented limits of detection, resolving power, and mass accuracy. As such, the Hicks lab has developed PepSAVI-MS (Statistically-guided bioActive Peptides prioritized Via Mass Spectrometry), an adaptable method leveraging mass spectrometry, sensitive bioactivity assays, and statistical analysis to rapidly identify AMPs in natural product extracts. Despite significant biochemical diversity, conserved features among AMPs can guide PepSAVI-MS identification of novel bioactive sequences. Antimicrobial peptides are often cysteine-rich; extensive disulfide formation among cysteine residues greatly enhances AMP stability and thus highlights the therapeutic attractiveness of this molecular class. Chemical derivatization strategies can be exploited to detect the presence of cysteine-rich peptides (CRPs) in complex matrices by mass shift analysis, where extracts before and after the reduction of disulfides and alkylation of cysteine residues produces a characteristic mass shift observable by LC-MS. Mass shift analysis can be leveraged to identify and prioritize putatively bioactive mass species in a sea of botanical macromolecules and rich secondary metabolites typically obscuring analyses (Chapters 2-5). Cyclotides are a class of head-to-tail cyclized, plant-derived CRPs with diverse and potent intrinsic bioactivities. Examination of Viola spp. via PepSAVI-MS and mass shift analysis has revealed new anticancer and antifungal bioactivities of a known cyclotide, cycloviolacin O8 (Chapter 3), and six novel antibacterial cyclotides, cycloviolacins I1-6 (Chapter 4). Elements of the discovery process can be modified to improve AMP identification, including alternative botanical tissue sources (Chapter 7), target pathogens (Chapter 3), mass spectral fragmentation techniques (Chapter 3), and bioactivity assay formats (Chapter 6). Furthermore, cyclic peptides recalcitrant to MS/MS fragmentation often challenge MS-based sequence characterization; as such, common cyclotide MS/MS “fingerprint” ions are determined and rapidly provide molecular information for novel cyclotide mass species prior to full sequence elucidation (Chapter 5). Herein, robust analytical methods support the exploration of complex botanical extracts and expand the biochemical repertoire of natural product AMPs.Doctor of Philosoph

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms

Biofilm formation is critical for the infection cycle of; Vibrio cholerae. Vibrio; exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for; V. cholerae; biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity

Polyketide synthase pathways identified from an arrayed metagenomic library are derived from soil Acidobacteria

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Polyketide synthase pathways identified from a metagenomic library are derived from soil Acidobacteria

Polyketides are structurally diverse secondary metabolites, many of which have antibiotic or anticancer activity. Type I modular polyketide synthase (PKS) genes are typically large and encode repeating enzymatic domains that elongate and modify the nascent polyketide chain. A fosmid metagenomic library constructed from an agricultural soil was arrayed and the macroarray was screened for the presence of conserved ketosynthase [β-ketoacyl synthase (KS)] domains, enzymatic domains present in PKSs. Thirty-four clones containing KS domains were identified by Southern hybridization. Many of the KS domains contained within metagenomic clones shared significant similarity to PKS or nonribosomal peptide synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. However, analysis of complete clone insert sequences indicated that the blast analysis for KS domains did not reflect the true phylogenetic origin of many of these metagenomic clones that had a %G+C content and significant sequence similarity to genes from members of the phylum Acidobacteria. This conclusion of an Acidobacteria origin for several clones was further supported by evidence that cultured soil Acidobacteria from different subdivisions have genetic loci closely related to PKS domains contained within metagenomic clones, suggesting that Acidobacteria may be a source of novel polyketides. This study also demonstrates the utility of combining data from culture-dependent and -independent investigations in expanding our collective knowledge of microbial genomic diversity. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd

Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C terminus.

The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK-BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK-BMAL1 to close the negative feedback loop and generate 24-h timing is not known. Here we show that, in mouse fibroblasts, CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK-BMAL1. TAD mutations that alter affinities for co-regulators affect the balance of repression and activation to consequently change the intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators, and they highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing

A Slow Conformational Switch in the BMAL1 Transactivation Domain Modulates Circadian Rhythms

The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms.

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms.

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity