Supramolecular assemblies in bacterial immunity: an emerging paradigm.

The study of bacterial immune systems has recently gained momentum, revealing a fascinating trend: many systems form large supramolecular assemblies. Here, we examine the potential mechanisms underpinning the evolutionary success of these structures, draw parallels to eukaryotic immunity, and offer fresh perspectives to stimulate future research into bacterial immunity.

Swapping the linkers of canonical Hsp70 and Hsp110 chaperones compromises both self-association and client selection.

Plasmodium falciparum heat shock protein 70-1 (PfHsp70-1) and PfHsp70-z are essential cytosol localised chaperones of the malaria parasite. The two chaperones functionally interact to drive folding of several parasite proteins. While PfHsp70-1 is regarded as a canonical Hsp70 chaperone, PfHsp70-z belongs to the Hsp110 subcluster. One of the distinctive features of PfHsp70-z is its unique linker segment which delineates it from canonical Hsp70. In the current study, we elucidated the role of the linker in regulating Hsp70 self-association and client selection. Using recombinant forms of PfHsp70-1, PfHsp70-z and E. coli Hsp70 (DnaK) and their respective linker switch mutants we investigated self-association of the chaperones using surface plasmon resonance (SPR) analysis. The effect of the changes on client selectivity was investigated on DnaK and its mutant through co-affinity chromatography coupled to LC-MS analysis. Our findings demonstrated that the linker is important for both Hsp70 self-association and client binding.

The major inducible small heat shock protein HSP20-3 in the tardigrade Ramazzottius varieornatus forms filament-like structures and is an active chaperone.

The tardigrade Ramazzottius varieornatus has remarkable resilience to a range of environmental stresses. In this study, we have characterised two members of the small heat shock protein (sHSP) family in R. varieornatus, HSP20-3 and HSP20-6. These are the most highly upregulated sHSPs in response to a 24 h heat shock at 35 0C of adult tardigrades with HSP20-3 being one of the most highly upregulated gene in the whole transcriptome. Both R. varieornatus sHSPs and the human sHSP, CRYAB (HSPB5), were produced recombinantly for comparative structure-function studies. HSP20-3 exhibited a superior chaperone activity than human CRYAB in a heat-induced protein aggregation assay. Both tardigrade sHSPs also formed larger oligomers than CRYAB as assessed by size exclusion chromatography and transmission electron microscopy of negatively stained samples. Whilst both HSP20-3 and HSP20-6 formed particles that were variable in size and larger than the particles formed by CRYAB, only HSP20-3 formed filament-like structures. The particles and filament-like structures formed by HSP20-3 appear inter-related as the filament-like structures often had particles located at their ends. Sequence analyses identified two unique features; an insertion in the middle region of the N-terminal domain (NTD) and preceding the critical-sequence identified in CRYAB, as well as a repeated QNTN-motif located in the C-terminal domain of HSP20-3. The NTD insertion is expected to affect protein-protein interactions and subunit oligomerisation. Removal of the repeated QNTN-motif abolished HSP20-3 chaperone activity and also affected the assembly of the filament-like structures. We discuss the potential contribution of HSP20-3 to protein condensate formation.

Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Modelling the Decamerisation Cycle of PRDX1 and the Inhibition-like Effect on Its Peroxidase Activity.

Peroxiredoxins play central roles in the detoxification of reactive oxygen species and have been modelled across multiple organisms using a variety of kinetic methods. However, the peroxiredoxin dimer-to-decamer transition has been underappreciated in these studies despite the 100-fold difference in activity between these forms. This is due to the lack of available kinetics and a theoretical framework for modelling this process. Using published isothermal titration calorimetry data, we obtained association and dissociation rate constants of 0.050 µM-4·s-1 and 0.055 s-1, respectively, for the dimer-decamer transition of human PRDX1. We developed an approach that greatly reduces the number of reactions and species needed to model the peroxiredoxin decamer oxidation cycle. Using these data, we simulated horse radish peroxidase competition and NADPH-oxidation linked assays and found that the dimer-decamer transition had an inhibition-like effect on peroxidase activity. Further, we incorporated this dimer-decamer topology and kinetics into a published and validated in vivo model of PRDX2 in the erythrocyte and found that it almost perfectly reconciled experimental and simulated responses of PRDX2 oxidation state to hydrogen peroxide insult. By accounting for the dimer-decamer transition of peroxiredoxins, we were able to resolve several discrepancies between experimental data and available kinetic models.

Cryo-EM structure of a DNA-PK trimer: higher order oligomerisation in NHEJ.

The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers.

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BACKGROUND: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. METHODS AND RESULTS: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. CONCLUSIONS: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix.

Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic.

Protein SUMOylation is a ubiquitylation-like post-translational modification (PTM) that is synthesized through an enzymatic cascade involving an E1 (SAE1:SAE2), an E2 (UBC9), and various E3 enzymes. In the final step of this process, the small ubiquitin-like modifier (SUMO) is transferred from the UBC9∼SUMO thioester onto a lysine residue of a protein substrate. This reaction can be accelerated by an E3 ligase. As the UBC9∼SUMO thioester is chemically unstable, a stable mimetic is desirable for structural studies of UBC9∼SUMO alone and in complex with a substrate and/or an E3 ligase. Recently, a strategy for generating a mimetic of the yeast E2∼SUMO thioester by mutating alanine 129 of Ubc9 to a lysine has been reported. Here, we reproduce and further investigate this approach using the human SUMOylation system and characterize the resulting mimetic of human UBC9∼SUMO1. We show that substituting lysine for alanine 129, but not for other active-site UBC9 residues, results in a UBC9 variant that is efficiently auto-SUMOylated. The auto-modification is dependent on cysteine 93 of UBC9, suggesting that it proceeds via this residue, through the same pathway as that for SUMOylation of substrates. The process is also partially dependent on aspartate 127 of UBC9 and accelerated by high pH, highlighting the importance of the substrate lysine protonation state for efficient SUMOylation. Finally, we present the crystal structure of the UBC9-SUMO1 molecule, which reveals the mimetic in an open conformation and its polymerization via the noncovalent SUMO-binding site on UBC9. Similar interactions could regulate UBC9∼SUMO in some cellular contexts.

Characterisation of a laccase isolated from Trametes hirsuta and its application in the oligomerisation of phenolic compounds.

Phenolic compounds are widely distributed in nature and industrial environment, and their detoxification or bioactive enhancement is of great value to environmental protection and industrial development. Laccases are multicopper oxidases that catalyse the oligo- or polymerisation of phenolic compounds. Identifying new laccase producers and investigating their application potential are of great importance. In this study, a white-rot fungus, Trametes hirsuta EZ1, with significantly high laccase productivity was isolated. The optimum conditions were studied for the maximum fermentation of extracellular laccase, which was achieved at 150 U/mL with a medium containing 10% strain EZ1, 7% maltodextrin, 1.5% peptone, and 0.5 mM Cu2+, and incubation at initial pH 6.0, 32 °C, and 180 rpm for nine days. Subsequently, a 70-kDa laccase was purified that showed activity over a wide range of temperature and pH, sensitivity to many metal ions and sodium dodecyl sulphate, and high tolerance to organic solvents. Purified laccase showed a significant unreported effect by catalysing catechol or ferulic acid into dimers, trimers, and tetramers or caffeic acid into dimers, trimers, tetramers, and pentamers. The oligomeric mixtures exhibited increased antioxidative capacity compared to that of each parent monomer, except for caffeic acid derivatives. Our study offers a novel strain source for laccase production and broadens its application in the enhancement of bioactive compounds.

The cryo-EM structure of full-length RAD52 protein contains an undecameric ring.

The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double-strand break repair. The N-terminal half of RAD52 is primarily responsible for self-oligomerisation and DNA binding. Crystallographic studies have revealed the detailed structure of the N-terminal half. However, only low-resolution structures have been reported for the full-length protein, and thus the structural role of the C-terminal half in self-oligomerisation has remained elusive. In this study, we determined the solution structure of the human RAD52 protein by cryo-electron microscopy (cryo-EM), at an average resolution of 3.5 Å. The structure revealed an undecameric ring that is nearly identical to the crystal structures of the N-terminal half. The cryo-EM map for the C-terminal half was poorly defined, indicating that the region is intrinsically disordered. The present cryo-EM structure provides important insights into the mechanistic roles played by the N-terminal and C-terminal halves of RAD52 during DNA double-strand break repair.

TRAF trimers form immune signalling networks via RING domain dimerization.

For many inflammatory cytokines, the response elicited is dependent on the recruitment of the tumour necrosis factor receptor-associated factor (TRAF) family of adaptor proteins. All TRAF proteins have a trimeric C-terminal TRAF domain, while at the N-terminus most TRAFs have a RING domain that forms dimers. The symmetry mismatch of the N- and C-terminal halves of TRAF proteins means that when receptors cluster, it is presumed that RING dimers connect TRAF trimers to form a network. Here, using purified TRAF6 proteins, we provide direct evidence in support of this model, and we show that TRAF6 trimers bind Lys63-linked ubiquitin chains to promote their processive assembly. This study provides critical evidence in support of TRAF trimers as key players in signalling.

Cloning, expression, purification and preliminary oligomerisation analysis of recombinant protein Mycobacterium tuberculosis Rv1288

Aims@#LysM containing-protein is widely distributed in all domains of life and this kind of protein is essential for various biological activities in living organisms. Rv1288, a LysM containing-protein with esterase, was found in Mycobacterium tuberculosis. Biophysical studies revealed that the protein is responsible for modulating lipid metabolism that enables pathogens to survive under extreme conditions and decrease the permeability of the pathogen’s cell wall to drug therapeutic agents. However, recognition and interaction between the protein, lipid and carbohydrate moieties at the molecular level remains largely unknown and must be investigated. Therefore, a production of recombinant protein Rv1288 should be performed to aid the study.@*Methodology and results@#In this study, we cloned the full-length cDNA of Rv1288 from M. tuberculosis strain H37Rv and expressed it in pET-24d- Escherichia coli BL21(DE3) cells. Affinity and size exclusion chromatography methods purified the protein, and its preliminary oligomerisation state was determined based on a calculated apparent molecular weight of the protein. Rv1288 was expressed as a soluble protein at 20 °C, induced with 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG). The calculated apparent molecular weight suggested that the Rv1288 protein formed a hexamer in solution. @*Conclusion, significance and impact of study@#All the methods involved in this study to produce the recombinant Rv1288-pET24d and its soluble protein in E. coli cells have been described. Hence, it can be implemented for future studies.

Mushroom Natural Products in Neurodegenerative Disease Drug Discovery.

The variety of drugs available to treat neurodegenerative diseases is limited. Most of these drug's efficacy is restricted by individual genetics and disease stages and usually do not prevent neurodegeneration acting long after irreversible damage has already occurred. Thus, drugs targeting the molecular mechanisms underlying subsequent neurodegeneration have the potential to negate symptom manifestation and subsequent neurodegeneration. Neuroinflammation is a common feature of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis, and is associated with the activation of the NLRP3 inflammasome, which in turn leads to neurodegeneration. Inflammasome activation and oligomerisation is suggested to be a major driver of disease progression occurring in microglia. With several natural products and natural product derivatives currently in clinical trials, mushrooms have been highlighted as a rich and largely untapped source of biologically active compounds in both in vitro and in vivo neurodegenerative disease models, partially supported by successful clinical trial evaluations. Additionally, novel high-throughput methods for the screening of natural product compound libraries are being developed to help accelerate the neurodegenerative disease drug discovery process, targeting neuroinflammation. However, the breadth of research relating to mushroom natural product high-throughput screening is limited, providing an exciting opportunity for further detailed investigations.

Diphosphorus Release and Heterocumulene Oligomerisation by Nickel Complexes.

The generation of diphosphorus molecules P2 under mild conditions in solution is a useful strategy to generate diphosphines via [4+2] cycloadditions. We recently described the release of P2 units from the nickel butterfly complex [{(IMes)Ni(CO)}2(µ2,η2:η2-P2)] (IMes=1,3-bis(2,4,6-trimethylphenyl)imidazolin-2-ylidene) upon addition of CO gas. Herein, we developed an alternative protocol for the same process using heterocumulenes. In addition to formation of P4 (the dimerisation product of P2), the reactions afford nickel complexes of novel pincer-type ligands. Aryl isothiocyanates undergo a trimerisation within the coordination sphere of nickel and afford square planar nickel complexes with S-C-S pincer-ligand frameworks. Carbon disulfide coordinates to the [(IMes)Ni]-fragment in an η2-fashion, affording a dinuclear complex. Similar products are formed when the N-heterocyclic carbene nickel(0) complex [(IMes)Ni(vtms)2] is used as a precursor (vtms=vinyltrimethylsilane).

AtSWEET11 and AtSWEET12: the twin traders of sucrose.

AtSWEET11 and AtSWEET12 are central players in phloem loading and long-distance sucrose translocation. During drought stress, these transporters enhance sucrose transport from shoot to root, increasing root proliferation. Chen et al. have now unravelled novel aspects of sucrose transport regulation, occurring via AtSWEET11 and AtSWEET12 phosphorylation and oligomerisation.

Adenosine receptor signalling in Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia in the elderly and its increasing prevalence presents treatment challenges. Despite a better understanding of the disease, the current mainstay of treatment cannot modify pathogenesis or effectively address the associated cognitive and memory deficits. Emerging evidence suggests adenosine G protein-coupled receptors (GPCRs) are promising therapeutic targets for Alzheimer's disease. The adenosine A1 and A2A receptors are expressed in the human brain and have a proposed involvement in the pathogenesis of dementia. Targeting these receptors preclinically can mitigate pathogenic ß-amyloid and tau neurotoxicity whilst improving cognition and memory. In this review, we provide an accessible summary of the literature on Alzheimer's disease and the therapeutic potential of A1 and A2A receptors. Although there are no available medicines targeting these receptors approved for treating dementia, we provide insights into some novel strategies, including allosterism and the targeting of oligomers, which may increase drug discovery success and enhance the therapeutic response.

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and ß-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.

Investigation of the Formation of Squalene Oligomers Exposed to Ultraviolet Light and Changes in the Exposed Squalene as a Potential Skin Model.

UV-induced oligomerisation of squalene was undertaken to indicate the potential for squalene-containing biological systems to exhibit rheology changes. DOSY NMR enabled the determination of the molecular weight (MW) range using Stokes-Einstein Gierer-Wirtz Estimation (SEGWE Calculator, University of Manchester). This approach was validated by Atmospheric Solids Analysis Probe Time of Flight Mass Spectrometry (ASAP TOF MS). To demonstrate the principle, both benzoyl peroxide and AIBN were used, separately, to initiate rapid, radical oligomerisation. Subsequent experiments in the absence of initiators compared the influence of UV wavelength and time on the resulting oligomer formation. To further model a relevant biological implication of this potentially chaotic UV oligomerisation, both saturated and unsaturated free fatty acids were added to squalene and exposed to UV at 285 nm and 300 nm to determine if cross oligomerisation could be observed. This representation of sebum evidenced the formation of a distribution of higher MW oligomers. Internal viscosity was normalised using the DMSO solvent, but to confirm that changes in rheology did not affect diffusion, a final experiment where fresh squalene was added to our oligomer mixture, representative of sebum, showed that unchanged squalene possessed the anticipated monomeric diffusion coefficient and hence MW. This work suggests, at least qualitatively, that UV-induced squalene oligomerisation can occur over time and that this may have a role in the behaviour of squalene on the skin.

A guide to the regulation of selective autophagy receptors.

Autophagy is a highly conserved catabolic process cells use to maintain their homeostasis by degrading misfolded, damaged and excessive proteins, nonfunctional organelles, foreign pathogens and other cellular components. Hence, autophagy can be nonselective, where bulky portions of the cytoplasm are degraded upon stress, or a highly selective process, where preselected cellular components are degraded. To distinguish between different cellular components, autophagy employs selective autophagy receptors, which will link the cargo to the autophagy machinery, thereby sequestering it in the autophagosome for its subsequent degradation in the lysosome. Autophagy receptors undergo post-translational and structural modifications to fulfil their role in autophagy, or upon executing their role, for their own degradation. We highlight the four most prominent protein modifications - phosphorylation, ubiquitination, acetylation and oligomerisation - that are essential for autophagy receptor recruitment, function and turnover. Understanding the regulation of selective autophagy receptors will provide deeper insights into the pathway and open up potential therapeutic avenues.

Structural Rearrangements of a Dodecameric Ketol-Acid Reductoisomerase Isolated from a Marine Thermophilic Methanogen.

Ketol-acid reductoisomerase (KARI) orchestrates the biosynthesis of branched-chain amino acids, an elementary reaction in prototrophic organisms as well as a valuable process in biotechnology. Bacterial KARIs belonging to class I organise as dimers or dodecamers and were intensively studied to understand their remarkable specificity towards NADH or NADPH, but also to develop antibiotics. Here, we present the first structural study on a KARI natively isolated from a methanogenic archaea. The dodecameric structure of 0.44-MDa was obtained in two different conformations, an open and close state refined to a resolution of 2.2-Å and 2.1-Å, respectively. These structures illustrate the conformational movement required for substrate and coenzyme binding. While the close state presents the complete NADP bound in front of a partially occupied Mg2+-site, the Mg2+-free open state contains a tartrate at the nicotinamide location and a bound NADP with the adenine-nicotinamide protruding out of the active site. Structural comparisons show a very high conservation of the active site environment and detailed analyses point towards few specific residues required for the dodecamerisation. These residues are not conserved in other dodecameric KARIs that stabilise their trimeric interface differently, suggesting that dodecamerisation, the cellular role of which is still unknown, might have occurred several times in the evolution of KARIs.