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Journal articleKomenda J, Sobotka R, Nixon PJ, 2024,
The biogenesis and maintenance of photosystem II: recent advances and current challenges
, The Plant Cell, Vol: 36, Pages: 3997-4013, ISSN: 1040-4651The growth of plants, algae and cyanobacteria relies on the catalytic activity of the oxygen-evolving photosystem two (PSII) complex which uses solar energy to extract electrons from water to feed into the photosynthetic electron transport chain. PSII is proving to be an excellent system to study how large multi-subunit membrane-protein complexes are assembled in the thylakoid membrane and subsequently repaired in response to photooxidative damage. Here we summarize recent developments in understanding the biogenesis of PSII, with an emphasis on recent insights obtained from biochemical and structural analysis of cyanobacterial PSII assembly/repair intermediates. We also discuss how chlorophyll synthesis is synchronized with protein synthesis and suggest a possible role for photosystem I in PSII assembly. Special attention is paid to unresolved and controversial issues that could be addressed in future research.
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Journal articleKviatkovski I, Zhong Q, Vaidya S, et al., 2024,
Identification of novel genetic factors that regulate c-di-AMP production in Staphylococcus aureus using a riboswitch-based biosensor
, mSphere, Vol: 9, ISSN: 2379-5042Nucleotide secondary messengers regulate various processes in bacteria allowing them to rapidly respond to changes in environmental conditions. c-di-AMP is an essential second messenger required for the growth of the human pathogen Staphylococcus aureus, regulating potassium, osmolyte uptake, and beta-lactam resistance. Cellular concentrations of c-di-AMP are regulated by the activities of two enzymes, DacA and GdpP, which synthesize and hydrolyze c-di-AMP, respectively. Besides these, only a limited number of other factors are known to regulate c-di-AMP levels. Using a c-di-AMP biosensor consisting of the Bacillus subtilis c-di-AMP-binding kimA riboswitch and yfp, we were able to efficiently detect differences in cellular c-di-AMP levels in S. aureus. To identify novel factors that regulate c-di-AMP levels, we introduced the biosensor into a library of S. aureus transposon mutants. In this manner, we obtained mutants with increased c-di-AMP levels that contained insertions in gdpP coding for the c-di-AMP hydrolase and ybbR (cdaR) coding for a c-di-AMP cyclase regulator, thus validating our screen. We also identified two high c-di-AMP mutants with insertions upstream of the nrdIEF operon coding for the ribonucleotide reductase enzyme. Further analysis revealed that the insertion down-regulated nrdIEF expression, indicating that the enzyme is a negative regulator of c-di-AMP production. This negative regulation was dependent on rsh, encoding for the synthase of the endogenous GdpP inhibitor (p)ppGpp. The methods established in this work can be readily adapted for use in other bacteria to uncover genetic or environmental factors regulating c-di-AMP levels.
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Journal articleGosiamemang T, Heng JYY, 2024,
Superhydrophobic cotton for addressing fatbergs through oily wastewater treatment
, International Journal of Biological Macromolecules, Vol: 277, ISSN: 0141-8130Fats, oils and grease (FOGs) deposits in sewers have recently become a significant problem, causing financial strain on water companies, damaging sewer lines, and exposing the environment to dirty water through sanitary sewer overflows. Despite the proactive use of grease traps/interceptors for physical oil-water separation, the issue of FOG deposits persists. This study proposes the use of adsorption-based oil-water separation, employing superhydrophobic cotton, as a new alternative method for removing FOGs. Durable superhydrophobic cotton was successfully prepared using a simple two-step sol-gel method, with octadecyltrimethoxysilane (ODTMS) as a modifying silane. The resulting cotton samples demonstrated remarkable superhydrophobicity, evidenced by water contact angle (WCA) above 154°. Additionally, it exhibited exceptional durability and stability when exposed to hot water, harsh acidic and alkaline solutions, as well as during a laundry test. Moreover, the cotton displayed excellent oil-water separation efficiency (> 98 %) and maintained consistent performance throughout 20 reuse cycles, highlighting its high reusability. This approach holds the potential to address the prevailing FOG deposit issues and contribute to more efficient and sustainable wastewater management practices.
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Journal articleSanchez Garrido J, Naemi Baghshomali Y, Kaushal P, et al., 2024,
Impaired neutrophil migration underpins host susceptibility to infectious colitis
, Mucosal Immunology, Vol: 17, Pages: 939-957, ISSN: 1933-0219Citrobacter rodentium models infection with enteropathogenic Escherichia coli and ulcerative colitis (UC). While C57BL/6 (C57) mice recover, C3H/HeN (C3H) mice succumb to infection, partially due to increased colonic neutrophil elastase activity, also seen in UC patients; however, the underlying cause was unknown. Here, we found that bone marrow, blood, and colonic C57 neutrophils expressed (CD)11bHi and reached the infected colonic lumen, where they underwent productive NETosis. In contrast, while the number of C3H neutrophils increased in the bone marrow, blood, and colon, they remained CD11bLo and got trapped in the submucosa, away from C. rodentium, where they underwent harmful NETosis. CD11bLo neutrophils in C3H mice infected with CRi9, which triggers expression of neutrophil chemoattractants, reached the colonization site, resulting in host survival. UC patient neutrophils also displayed decreased levels of the activation/differentiation markers CD16/CXCR4. These results, suggesting that neutrophil malfunction contributes to exacerbated colitis, provide insight for future therapeutic prospects.
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Journal articleChen W, Park Y-K, Studená L, et al., 2024,
Synthetic, marine, light-driven, autotroph-heterotroph co-culture system for sustainable β-caryophyllene production
, Bioresource Technology, Vol: 410, ISSN: 0960-8524Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO2 and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium Synechococcus sp. PCC 7002 and an industrial yeast Yarrowia lipolytica. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, Yarrowia lipolytica was engineered to efficiently use sucrose and produce β-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum β-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and Y. lipolytica, and provides a foundation for sustainable bioproduction from CO2 and light through co-culture systems.
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Journal articleYuen ELH, Tumtas Y, King F, et al., 2024,
A pathogen effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion
, Science Advances, Vol: 10, ISSN: 2375-2548Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant-derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TOPGAP, to remodel the host-pathogen interface. The effector, PiE354, hijacks TOPGAP as a susceptibility factor to usurp its GAP activity on Rab8a, a key Rab GTPase crucial for defense-related secretion. By hijacking TOPGAP, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co-opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.
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Journal articleRothe P, Wamp S, Rismondo J, et al., 2024,
Cytosolic factors controlling PASTA kinase-dependent ReoM phosphorylation
, Molecular Microbiology, Vol: 122, Pages: 514-533, ISSN: 0950-382XBacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram-positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram-positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of in vivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth phase-dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must be able to bind its substrates and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases were not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation.
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Journal articleJiang L, Ning Z, Gong Z, et al., 2024,
Unveiling Crystal Structure Landscapes and Morphology Modification Rules of Substituted Benzophenone Crystals
, CRYSTAL GROWTH & DESIGN, Vol: 24, Pages: 8476-8487, ISSN: 1528-7483 -
Journal articleHutchison CDM, Perrett S, van Thor JJ, 2024,
XFEL beamline optical instrumentation for ultrafast science
, The Journal of Physical Chemistry B, Vol: 128, Pages: 8855-8868, ISSN: 1520-6106Free electron lasers operating in the soft and hard X-ray regime provide capabilities for ultrafast science in many areas, including X-ray spectroscopy, diffractive imaging, solution and material scattering, and X-ray crystallography. Ultrafast time-resolved applications in the picosecond, femtosecond, and attosecond regimes are often possible using single-shot experimental configurations. Aside from X-ray pump and X-ray probe measurements, all other types of ultrafast experiments require the synchronized operation of pulsed laser excitation for resonant or nonresonant pumping. This Perspective focuses on the opportunities for the optical control of structural dynamics by applying techniques from nonlinear spectroscopy to ultrafast X-ray experiments. This typically requires the synthesis of two or more optical pulses with full control of pulse and interpulse parameters. To this end, full characterization of the femtosecond optical pulses is also highly desirable. It has recently been shown that two-color and two-pulse femtosecond excitation of fluorescent protein crystals allowed a Tannor-Rice coherent control experiment, performed under characterized conditions. Pulse shaping and the ability to synthesize multicolor and multipulse conditions are highly desirable and would enable XFEL facilities to offer capabilities for structural dynamics. This Perspective will give a summary of examples of the types of experiments that could be achieved, and it will additionally summarize the laser, pulse shaping, and characterization that would be recommended as standard equipment for time-resolved XFEL beamlines, with an emphasis on ultrafast time-resolved serial femtosecond crystallography.
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Journal articleCioccolo S, Barritt JD, Pollock N, et al., 2024,
The mycobacterium lipid transporter MmpL3 is dimeric in detergent solution, SMALPs and reconstituted nanodiscs
, RSC Chemical Biology, Vol: 5, Pages: 901-913, ISSN: 2633-0679The mycobacterial membrane protein large 3 (MmpL3) transports key precursor lipids to the outer membrane of Mycobacterium species. Multiple structures of MmpL3 from both M. tuberculosis and M. smegmatis in various conformational states indicate that the protein is both structurally and functionally monomeric. However, most other resistance, nodulation and cell division (RND) transporters structurally characterised to date are either dimeric or trimeric. Here we present an in depth biophysical and computational analysis revealing that MmpL3 from M. smegmatis exists as a dimer in a variety of membrane mimetic systems (SMALPs, detergent-based solution and nanodiscs). Sucrose gradient separation of MmpL3 populations from M. smegmatis, reconstituted into nanodiscs, identified monomeric and dimeric populations of the protein using laser induced liquid bead ion desorption (LILBID), a native mass spectrometry technique. Preliminary cryo-EM analysis confirmed that MmpL3 forms physiological dimers. Untargeted lipidomics experiments on membrane protein co-purified lipids revealed PE and PG lipid classes were predominant. Molecular dynamics simulations, in the presence of physiologically-relevant lipid compositions revealed the likely dimer interface.
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Conference paperMazza T, Roumeliotis TI, Garitta E, et al., 2024,
Unveiling the Modulation of MRP2 Activity: Insights from Phosphorylation and Drug Interactions
, 22nd European Bioenergetics Conference (EBEC), Publisher: ELSEVIER, Pages: 84-85, ISSN: 0005-2728 -
Conference paperZhao Z, Vercellino I, Nixon PJ, et al., 2024,
Structural analysis of dimeric photosystem II complexes
, 22nd European Bioenergetics Conference (EBEC), Publisher: ELSEVIER, Pages: 52-52, ISSN: 0005-2728 -
Journal articleVega-Benedetti AF, Porcedda C, Ercoli T, et al., 2024,
Immune responses to oligomeric α-synuclein in Parkinson's disease peripheral blood mononuclear cells
, JOURNAL OF NEUROLOGY, Vol: 271, Pages: 5916-5929, ISSN: 0340-5354 -
Journal articleLingche H, Miguel-Romero L, Patkowski JB, et al., 2024,
Tail assembly interference is a common strategy in bacterial antiviral defenses
, Nature Communications, Vol: 15, ISSN: 2041-1723Many bacterial immune systems recognize phage structural components to activate antiviral responses, without inhibiting the function of the phage component. These systems can be encoded in specific chromosomal loci, known as defense islands, and in mobile genetic elements such as prophages and phage-inducible chromosomal islands (PICIs). Here, we identify a family of bacterial immune systems, named Tai (for ‘tail assembly inhibition’), that is prevalent in PICIs, prophages and P4-like phage satellites. Tai systems protect their bacterial host population from other phages by blocking the tail assembly step, leading to the release of tailless phages incapable of infecting new hosts. To prevent autoimmunity, some Tai-positive phages have an associated counter-defense mechanism that is expressed during the phage lytic cycle and allows for tail formation. Interestingly, the Tai defense and counter-defense genes are organized in a non-contiguous operon, enabling their coordinated expression.
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Journal articleReuter LM, Khadayate SP, Mossler A, et al., 2024,
MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing
, Nature Communications, Vol: 15, ISSN: 2041-1723Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.
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Journal articleEsposito A, Leone L, De Simone A, et al., 2024,
Catalytic Nanomaterials by Conjugation of an Artificial Heme-Peroxidase to Amyloid Fibrils
, ACS APPLIED MATERIALS & INTERFACES, Vol: 16, Pages: 45371-45382, ISSN: 1944-8244 -
Journal articleFelipe-Ruiz A, Zamora-Caballero S, Bendori SO, et al., 2024,
Extracellular proteolysis of tandemly duplicated pheromone propeptides affords additional complexity to bacterial quorum sensing
, PLoS Biology, Vol: 22, ISSN: 1544-9173Bacterial interactions are vital for adapting to changing environments, with quorum sensing (QS) systems playing a central role in coordinating behaviors through small signaling molecules. The RRNPPA family is the prevalent QS systems in Bacillota and mediating communication through secreted oligopeptides, which are processed into active pheromones by extracellular proteases. Notably, in several cases the propeptides show the presence of multiple putative pheromones within their sequences, which has been proposed as a mechanism to diversify peptide-receptor specificity and potentially facilitate new functions. However, neither the processes governing the maturation of propeptides containing multiple pheromones, nor their functional significance has been evaluated. Here, using 2 Rap systems from bacteriophages infecting Bacillus subtilis that exhibit different types of pheromone duplication in their propeptides, we investigate the maturation process and the molecular and functional activities of the produced pheromones. Our results reveal that distinct maturation processes generate multiple mature pheromones, which bind to receptors with varying affinities but produce identical structural and biological responses. These findings add additional layers in the complexity of QS communication and regulation, opening new possibilities for microbial social behaviors, highlighting the intricate nature of bacterial interactions and adaptation.
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Journal articleDel Regno R, Della Sala P, Santonoceta GDG, et al., 2024,
Under the Influence of Water: Molecular Recognition of Organic Hydrophilic Molecules in Water with a Prismarene Host Driven by Hydration Effects
, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 30, ISSN: 0947-6539 -
Journal articleHohl M, Banks E, Manley M, et al., 2024,
Bidirectional pilus processing in the Tad pilus system motor CpaF
, Nature Communications, Vol: 15, ISSN: 2041-1723The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.
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Journal articleKabasakal BV, McFarlane CR, Cotton CAR, et al., 2024,
The crystal structure of Shethna protein II (FeSII) from Azotobacter vinelandii suggests a domain swap
, Acta Crystallographica Section D: Structural Biology, Vol: 80, Pages: 599-604, ISSN: 2059-7983The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239–247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe–2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe–2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.
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Journal articleAl Musaimi O, Ng KW, Gavva V, et al., 2024,
Elastin-derived peptide-based hydrogels as a potential drug delivery system
, Gels, Vol: 10, ISSN: 2310-2861A peptide-based hydrogel sequence was computationally predicted from the Ala-rich cross-linked domains of elastin. Three candidate peptides were subsequently synthesised and characterised as potential drug delivery vehicles. The elastin-derived peptides are Fmoc-FFAAAAKAA-NH2, Fmoc-FFAAAKAA-NH2, and Fmoc-FFAAAKAAA-NH2. All three peptide sequences were able to self-assemble into nanofibers. However, only the first two could form hydrogels, which are preferred as delivery systems compared to solutions. Both of these peptides also exhibited favourable nanofiber lengths of at least 1.86 and 4.57 µm, respectively, which are beneficial for the successful delivery and stability of drugs. The shorter fibre lengths of the third peptide (maximum 0.649 µm) could have inhibited their self-assembly into the three-dimensional networks crucial to hydrogel formation.
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Journal articleWolput S, Lood C, Fillol-Salom A, et al., 2024,
Phage-host co-evolution has led to distinct generalized transduction strategies
, Nucleic Acids Research, Vol: 52, Pages: 7780-7791, ISSN: 0305-1048Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction.
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Journal articleYu X-J, Xie H, Li Y, et al., 2024,
Modulation of Salmonella virulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex
, mBio, Vol: 15, ISSN: 2150-7511The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterise the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis, that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed between the five subspecies of Salmonella enterica, is found in many clinically-relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. Translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely -2-syntrophin, utrophin/ dystrophin, -catulin, -dystrobrevin and -dystrobrevin. The interaction between SseM and -2-syntrophin and -dystrobrevin was verified in S. Typhimurium-infected cells and relied on the PDZ domain of -2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A sseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates systemic growth of bacteria in mice. Furthermore, the sseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence.
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Journal articleBade I, Karde V, Schenck L, et al., 2024,
Process-induced crystal surface anisotropy and the impact on the powder properties of Odanacatib
, Pharmaceutics, Vol: 16, ISSN: 1999-4923Crystalline active pharmaceutical ingredients with comparable size and surface area can demonstrate surface anisotropy induced during crystallization or downstream unit operations such as milling. To the extent that varying surface properties impacts bulk powder properties, the final drug product performance such as stability, dissolution rates, flowability, and dispersibility can be predicted by understanding surface properties such as surface chemistry, energetics, and wettability. Here, we investigate the surface properties of different batches of Odanacatib prepared through either jet milling or fast precipitation from various solvent systems, all of which meet the particle size specification established to ensure equivalent biopharmaceutical performance. This work highlights the use of orthogonal surface techniques such as Inverse Gas Chromatography (IGC), Brunauer–Emmett–Teller (BET) surface area, contact angle, and X-ray Photoelectron Spectroscopy (XPS) to demonstrate the effect of processing history on particle surface properties to explain differences in bulk powder properties.
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Journal articleLi Z, Di Vagno L, Chawla H, et al., 2024,
Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells.
, bioRxivMammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.
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Journal articlePirnay J-P, Van Bambeke F, Baysse C, et al., 2024,
In memoriam: Pierre Cornelis (1949-2023)
, JOURNAL OF BACTERIOLOGY, ISSN: 0021-9193 -
Journal articleBaxter JM, Hutchison CDM, Fadini A, et al., 2024,
Power Density Titration of Reversible Photoisomerization of a Fluorescent Protein Chromophore in the Presence of Thermally Driven Barrier Crossing Shown by Quantitative Millisecond Serial Synchrotron X-ray Crystallography
, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 146, Pages: 16394-16403, ISSN: 0002-7863 -
Journal articleLane BJ, Ma Y, Yan N, et al., 2024,
Monitoring the conformational ensemble and lipid environment of a mechanosensitive channel under cyclodextrin-induced membrane tension
, STRUCTURE, Vol: 32, ISSN: 0969-2126 -
Journal articleCawood EE, Baker E, Edwards TA, et al., 2024,
Understanding β-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification
, CHEMICAL SCIENCE, ISSN: 2041-6520 -
Journal articleKalemera MD, Maher AK, Dominguez-Villar M, et al., 2024,
Cell culture evaluation hints widely available HIV drugs are primed for success if repurposed for HTLV-1 prevention
, Pharmaceuticals, Vol: 17, ISSN: 1424-8247With an estimated 10 million people infected, the deltaretrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is the second most prevalent pathogenic retrovirus in humans after HIV-1. Like HIV-1, HTLV-1 overwhelmingly persists in a host via a reservoir of latently infected CD4+ T cells. Although most patients are asymptomatic, HTLV-1-associated pathologies are often debilitating and include adult T-cell leukaemia/lymphoma (ATLL), which presents in mature adulthood and is associated with poor prognosis with short overall survival despite treatment. Curiously, the strongest indicator for the development of ATLL is the acquisition of HTLV-1 through breastfeeding. There are no therapeutic or preventative regimens for HTLV-1. However, antiretrovirals (ARVs), which target the essential retrovirus enzymes, have been developed for and transformed HIV therapy. As the architectures of retroviral enzyme active sites are highly conserved, some HIV-specific compounds are active against HTLV-1. Here, we expand on our work, which showed that integrase strand transfer inhibitors (INSTIs) and some nucleoside reverse transcriptase inhibitors (NRTIs) block HTLV-1 transmission in cell culture. Specifically, we find that dolutegravir, the INSTI currently recommended as the basis of all new combination antiretroviral therapy prescriptions, and the latest prodrug formula of the NRTI tenofovir, tenofovir alafenamide, also potently inhibit HTLV-1 infection. Our results, if replicated in a clinical setting, could see transmission rates of HTLV-1 and future caseloads of HTLV-1-associated pathologies like ATLL dramatically cut via the simple repurposing of already widely available HIV pills in HTLV-1 endemic areas. Considering our findings with the old medical saying “it is better to prevent than cure”, we highly recommend the inclusion of INSTIs and tenofovir prodrugs in upcoming HTLV-1 clinical trials as potential prophylactics.
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