Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models.

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered "Crab"-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. "Crab"-like configuration of two catalytic peptides ("pincers") flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for "single-pincer" conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled "Crabs" to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5'-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

Utilising Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to track the oxidation of lignin by an alkaliphilic laccase.

Lignin is a complex heteroaromatic polymer which is one of the most abundant and diverse biopolymers on the planet. It comprises approximately one third of all woody plant matter, making it an attractive candidate as an alternative, renewable feedstock to petrochemicals to produce fine chemicals. However, the inherent complexity of lignin makes it difficult to analyse and characterise using common analytical techniques, proving a hindrance to the utilisation of lignin as a green chemical feedstock. Herein we outline the tracking of lignin degradation by an alkaliphilic laccase in a semi-quantitative manner using a combined chemical analysis approach using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to characterise shifts in chemical diversity and relative abundance of ions, and NMR to highlight changes in the structure of lignin. Specifically, an alkaliphilic laccase was used to degrade an industrially relevant lignin, with compounds such as syringaresinol being almost wholly removed (95%) after 24 hours of treatment. Structural analyses reinforced these findings, indicating a >50% loss of NMR signal relating to ß-ß linkages, of which syringaresinol is representative. Ultimately, this work underlines a combined analytical approach that can be used to gain a broader semi-quantitative understanding of the enzymatic activity of laccases within a complex, non-model mixture.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Two-color coincidence single-molecule pulldown for the specific detection of disease-associated protein aggregates.

Protein misfolding and aggregation is a characteristic of many neurodegenerative disorders, including Alzheimer's and Parkinson's disease. The oligomers generated during aggregation are likely involved in disease pathogenesis and present promising biomarker candidates. However, owing to their small size and low concentration, specific tools to quantify and characterize aggregates in complex biological samples are still lacking. Here, we present single-molecule two-color aggregate pulldown (STAPull), which overcomes this challenge by probing immobilized proteins using orthogonally labeled detection antibodies. By analyzing colocalized signals, we can eliminate monomeric protein and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 picomolar. Furthermore, we show that STAPull can be used in a range of samples, including human biofluids. STAPull is applicable to protein aggregates from a variety of disorders and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.

3'-O-ß-Glucosyl-4',5'-didehydro-5'-deoxyadenosine Is a Natural Product of the Nucleocidin Producers Streptomyces virens and Streptomyces calvus.

3'-O-ß-Glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 is identified as a natural product of Streptomyces calvus and Streptomyces virens. It is also generated in vitro by direct ß-glucosylation of 4',5'-didehydro-5'-deoxyadenosine 12 with the enzyme NucGT. The intact incorporation of oxygen-18 and deuterium isotopes from (±)[1-18O,1-2H2]-glycerol 14 into C-5' of nucleocidin 1 and its related metabolites precludes 3'-O-ß-glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 as a biosynthetic precursor to nucleocidin 1.

Intervention to reduce sedentary behaviour and improve outcomes after stroke (Get Set Go): a study protocol for the process evaluation of a pilot cluster randomised controlled trial (RECREATE).

INTRODUCTION: Stroke survivors spend long periods of time engaging in sedentary behaviour (SB) even when their functional recovery is good. In the RECREATE programme, an intervention aimed at reducing SB ('Get Set Go') will be implemented and evaluated in a pragmatic external pilot cluster randomised controlled trial with embedded process and economic evaluations. We report the protocol for the process evaluation which will address the following objectives: (1) describe and clarify causal assumptions about the intervention, and its mechanisms of impact; (2) assess implementation fidelity; (3) explore views, perceptions and acceptability of the intervention to staff, stroke survivors and their carers; (4) establish the contextual factors that influence implementation, intervention mechanisms and outcomes. METHODS AND ANALYSIS: This pilot trial will be conducted in 15 UK-based National Health Service stroke services. This process evaluation study, underpinned by the Medical Research Council guidance, will be undertaken in six of the randomised services (four intervention, two control). Data collection includes the following: observations of staff training sessions, non-participant observations in inpatient and community settings, semi-structured interviews with staff, patients and carers, and documentary analysis of key intervention components. Additional quantitative implementation data will be collected in all sites. Training observations and documentary analysis data will be summarised, with other observational and interview data analysed using thematic analysis. Relevant theories will be used to interpret the findings, including the theoretical domains framework, normalisation process theory and the theoretical framework of acceptability. Anticipated outputs include the following: recommendations for intervention refinements (both content and implementation); a revised implementation plan and a refined logic model. ETHICS AND DISSEMINATION: The study was approved by Yorkshire & The Humber - Bradford Leeds Research Ethics Committee (REC reference: 19/YH/0403). Findings will be disseminated via peer review publications, and national and international conference presentations. TRIAL REGISTRATION NUMBER: ISRCTN82280581.

Purification and biochemical characterization of SM14est, a PET-hydrolyzing enzyme from the marine sponge-derived Streptomyces sp. SM14.

The successful enzymatic degradation of polyester substrates has fueled worldwide investigation into the treatment of plastic waste using bio-based processes. Within this realm, marine-associated microorganisms have emerged as a promising source of polyester-degrading enzymes. In this work, we describe the hydrolysis of the synthetic polymer PET by SM14est, a polyesterase which was previously identified from Streptomyces sp. SM14, an isolate of the marine sponge Haliclona simulans. The PET hydrolase activity of purified SM14est was assessed using a suspension-based assay and subsequent analysis of reaction products by UV-spectrophotometry and RP-HPLC. SM14est displayed a preference for high salt conditions, with activity significantly increasing at sodium chloride concentrations from 100 mM up to 1,000 mM. The initial rate of PET hydrolysis by SM14est was determined to be 0.004 s-1 at 45°C, which was increased by 5-fold to 0.02 s-1 upon addition of 500 mM sodium chloride. Sequence alignment and structural comparison with known PET hydrolases, including the marine halophile PET6, and the highly efficient, thermophilic PHL7, revealed conserved features of interest. Based on this work, SM14est emerges as a useful enzyme that is more similar to key players in the area of PET hydrolysis, like PHL7 and IsPETase, than it is to its marine counterparts. Salt-tolerant polyesterases such as SM14est are potentially valuable in the biological degradation of plastic particles that readily contaminate marine ecosystems and industrial wastewaters.

Lipidomic Analysis Reveals Differences in Bacteroides Species Driven Largely by Plasmalogens, Glycerophosphoinositols and Certain Sphingolipids.

There has been increasing interest in bacterial lipids in recent years due, in part, to their emerging role as molecular signalling molecules. Bacteroides thetaiotaomicron is an important member of the mammalian gut microbiota that has been shown to produce sphingolipids (SP) that pass through the gut epithelial barrier to impact host SP metabolism and signal into host inflammation pathways. B. thetaiotaomicron also produces a novel family of N-acyl amines (called glycine lipids) that are potent ligands of host Toll-like receptor 2 (TLR2). Here, we specifically examine the lipid signatures of four species of gut-associated Bacteroides. In total we identify 170 different lipids, and we report that the range and diversity of Bacteroides lipids is species specific. Multivariate analysis reveals that the differences in the lipid signatures are largely driven by the presence and absence of plasmalogens, glycerophosphoinositols and certain SP. Moreover, we show that, in B. thetaiotaomicron, mutations altering either SP or glycine lipid biosynthesis result in significant changes in the levels of other lipids, suggesting the existence of a compensatory mechanisms required to maintain the functionality of the bacterial membrane.

Design and Fabrication of a Fully-Integrated, Miniaturised Fluidic System for the Analysis of Enzyme Kinetics.

The lab-on-a-chip concept, enabled by microfluidic technology, promises the integration of multiple discrete laboratory techniques into a miniaturised system. Research into microfluidics has generally focused on the development of individual elements of the total system (often with relatively limited functionality), without full consideration for integration into a complete fully optimised and miniaturised system. Typically, the operation of many of the reported lab-on-a-chip devices is dependent on the support of a laboratory framework. In this paper, a demonstrator platform for routine laboratory analysis is designed and built, which fully integrates a number of technologies into a single device with multiple domains such as fluidics, electronics, pneumatics, hydraulics, and photonics. This facilitates the delivery of breakthroughs in research, by incorporating all physical requirements into a single device. To highlight this proposed approach, this demonstrator microsystem acts as a fully integrated biochemical assay reaction system. The resulting design determines enzyme kinetics in an automated process and combines reservoirs, three-dimensional fluidic channels, optical sensing, and electronics in a low-cost, low-power and portable package.

Determining the Location of the α-Synuclein Dimer Interface Using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry.

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson's disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn.

Membrane lipids from gut microbiome-associated bacteria as structural and signalling molecules.

Bacteria produce an array of diverse, dynamic and often complex lipid structures, some of which function beyond their typical role in membrane structure. The model organism, E. coli, has three major membrane lipids, which are glycerophosphoglycerol (phosphatidylglycerol), glycerophosphoethanolamine (phosphatidylethanolamine) and cardiolipin. However, it is now appreciated that some bacteria have the capacity to synthesize a range of lipids, including glycerophosphocholines, glycerophosphoinositols, 'phosphorous-free' N-acyl amines, sphingolipids and plasmalogens. In recent years, some of these bacterial lipids have emerged as influential contributors to the microbe-host molecular dialogue. This review outlines our current knowledge of bacterial lipid diversity, with a focus on the membrane lipids of microbiome-associated bacteria that have documented roles as signalling molecules.

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

A mechanistic evaluation of human beta defensin 2 mediated protection of human skin barrier in vitro.

The human skin barrier, a biological imperative, is impaired in inflammatory skin diseases such as atopic dermatitis (AD). Staphylococcus aureus is associated with AD lesions and contributes to pathological inflammation and further barrier impairment. S. aureus secretes extracellular proteases, such as V8 (or 'SspA'), which cleave extracellular proteins to reduce skin barrier. Previous studies demonstrated that the host defence peptide human beta-defensin 2 (HBD2) prevented V8-mediated damage. Here, the mechanism of HBD2-mediated barrier protection in vitro is examined. Application of exogenous HBD2 provided protection against V8, irrespective of timeline of application or native peptide folding, raising the prospect of simple peptide analogues as therapeutics. HBD2 treatment, in context of V8-mediated damage, modulated the proteomic/secretomic profiles of HaCaT cells, altering levels of specific extracellular matrix proteins, potentially recovering V8 damage. However, HBD2 alone did not substantially modulate cellular proteomic/secretomics profiles in the absence of damage, suggesting possible therapeutic targeting of lesion damage sites only. HBD2 did not show any direct protease inhibition or induce expression of known antiproteases, did not alter keratinocyte migration or proliferation, or form protective nanonet structures. These data validate the barrier-protective properties of HBD2 in vitro and establish key protein datasets for further targeted mechanistic analyses.

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson's disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)-derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

A ribosomally synthesised and post-translationally modified peptide containing a ß-enamino acid and a macrocyclic motif.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally complex natural products with diverse bioactivities. Here we report discovery of a RiPP, kintamdin, for which the structure is determined through spectroscopy, spectrometry and genomic analysis to feature a bis-thioether macrocyclic ring and a ß-enamino acid residue. Biosynthetic investigation demonstrated that its pathway relies on four dedicated proteins: phosphotransferase KinD, Lyase KinC, kinase homolog KinH and flavoprotein KinI, which share low homologues to enzymes known in other RiPP biosynthesis. During the posttranslational modifications, KinCD is responsible for the formation of the characteristic dehydroamino acid residues including the ß-enamino acid residue, followed by oxidative decarboxylation on the C-terminal Cys and subsequent cyclization to provide the bis-thioether ring moiety mediated by coordinated action of KinH and KinI. Finally, conserved genomic investigation allows further identification of two kintamdin-like peptides among the kin-like BGCs, suggesting the occurrence of RiPPs from actinobacteria.

Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition.

Contemporary medicinal chemistry considers fragment-based drug discovery (FBDD) and inhibition of protein-protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano-ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow-up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C-terminal domain. An in-silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose-dependent inhibitor of the target PPI.

Probing TDP-43 condensation using an in silico designed aptamer.

Aptamers are artificial oligonucleotides binding to specific molecular targets. They have a promising role in therapeutics and diagnostics but are often difficult to design. Here, we exploited the catRAPID algorithm to generate aptamers targeting TAR DNA-binding protein 43 (TDP-43), whose aggregation is associated with Amyotrophic Lateral Sclerosis. On the pathway to forming insoluble inclusions, TDP-43 adopts a heterogeneous population of assemblies, many smaller than the diffraction-limit of light. We demonstrated that our aptamers bind TDP-43 and used the tightest interactor, Apt-1, as a probe to visualize TDP-43 condensates with super-resolution microscopy. At a resolution of 10 nanometers, we tracked TDP-43 oligomers undetectable by standard approaches. In cells, Apt-1 interacts with both diffuse and condensed forms of TDP-43, indicating that Apt-1 can be exploited to follow TDP-43 phase transition. The de novo generation of aptamers and their use for microscopy opens a new page to study protein condensation.