RESUMO
Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.
Assuntos
Inflamação/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Mitocôndrias/enzimologia , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Ciclo do Ácido Cítrico , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Malonatos/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Succinato Desidrogenase/genética , TranscriptomaRESUMO
Real-time tracking of prodrug uptake, delivery and activation inâ vivo represents a major challenge for prodrug development. Herein, we demonstrate the use of novel glycosylated theranostics of the cancer pharmacophore Amonafide in highly-selective, enzymatic triggered release. We show that the use of endogenous enzymes for activated release of the therapeutic component can be observed, in real time, and monitored using one and two-photon bioimaging, offering unique insight into the prodrug pharmacokinetic profile. Furthermore, we demonstrate that the potent cytotoxicity of Amonafide is preserved using this targeted approach.
Assuntos
Neoplasias , Pró-Fármacos , Humanos , Medicina de Precisão , Pró-Fármacos/farmacologia , Nanomedicina TeranósticaRESUMO
A unique V-shaped "chiral" supramolecular scaffold, N-(4-pyridyl)-4-amino-1,8-naphthalimide Tröger's base (TBNap), was synthesized in good yield from a precursor N-(4-pyridyl)-4-amino-1,8-naphthalimide (Nap). TBNap was characterized using different spectroscopic methods and the molecular structure was elucidated by diffraction analysis. A new p-cymene-Ru(II)-curcumin conjugate (TB-Ru-Cur) was designed by reacting TBNap dipyridyl donor and ruthenium-curcuminato acceptor [RuCur = (p-cymene)Ru-(curcuminato)Cl] in the presence of silver triflate. TB-Ru-Cur was isolated in quantitative yield and characterized using Fourier transform infrared (FT-IR), NMR (1H, 13C, and 19F), and electrospray ionization mass spectrometry (ESI-MS), and the molecular structure has been predicted using a computational study. Both TBNap and TB-Ru-Cur exhibited intramolecular charge transfer (ICT)-based fluorescence emission. Furthermore, the anticancer properties of TBNap, Ru-Cur, and TB-Ru-Cur were assessed in different cancer cell lines. Gratifyingly, the conjugate TB-Ru-Cur displayed fast-cellular internalization and good cytotoxicity against HeLa, HCT-116, and HepG2 cancer cells and the estimated IC50 value was much lower than that of the precursors (TBNap and Ru-Cur) and the well-known chemotherapeutic drug cisplatin.
Assuntos
Antineoplásicos , Complexos de Coordenação , Curcumina , Rutênio , 1-Naftilamina/análogos & derivados , Antineoplásicos/química , Linhagem Celular Tumoral , Complexos de Coordenação/química , Curcumina/química , Curcumina/farmacologia , Cimenos , Humanos , Naftalimidas , Quinolonas , Rutênio/química , Rutênio/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
An investigation into the self-assembly of two 4-amino- and a 4-bromo-1,8-naphthalimide (Nap) based structures (1-3) possessing an appended glycan unit, from protic polar media, is presented. The results demonstrate the formation of complex hierarchical luminescent aggregates, wherein the morphologies, sizes and spherical structures were highly dependent on both the media and the Nap structure. Upon cleaving the native glycosidic bond, using an enzyme, the structure/morphology of the self-assembly of 3 in buffered solution was significantly transformed.
RESUMO
The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.
Assuntos
Cerveja/microbiologia , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Membrana Celular/metabolismo , Cromossomos Fúngicos/genética , Evolução Molecular , Proteínas Fúngicas/metabolismo , Deleção de Genes , Expressão Gênica , Transferência Genética Horizontal , Genoma Fúngico/genética , Fases de Leitura Aberta , Saccharomyces/classificação , Saccharomyces/genética , Saccharomyces/crescimento & desenvolvimento , Saccharomyces/isolamento & purificação , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The inflammatory effects of carbon nanoparticles (NPs) are highly disputed. Here it is demonstrated that endotoxin-free preparations of raw carbon nanotubes (CNTs) are very limited in their capacity to promote inflammatory responses in vitro, as well as in vivo. Upon purification and selective oxidation of raw CNTs, a higher dispersibility is achieved in physiological solutions, but this process also enhances their inflammatory activity. In synergy with toll-like receptor (TLR) ligands, CNTs promote NLRP3 inflammasome activation and it is shown for the first time that this property extends to spherical carbon nano-onions (CNOs) of 6 nm in size. In contrast, the benzoic acid functionalization of purified CNTs and CNOs leads to significantly attenuated inflammatory properties. This is evidenced by a reduced secretion of the inflammatory cytokine IL-1ß, and a pronounced decrease in the recruitment of neutrophils and monocytes following injection into mice. Collectively, these results reveal that the inflammatory properties of carbon NPs are highly dependent on their physicochemical characteristics and crucially, that chemical surface functionalization allows significant moderation of these properties.
Assuntos
Proteínas de Transporte/química , Inflamação/patologia , Nanotubos de Carbono/química , Animais , Células Apresentadoras de Antígenos , Caspase 1/metabolismo , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Ligantes , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nanopartículas/química , Nanotecnologia , Oxigênio/química , Propriedades de SuperfícieRESUMO
Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli, and S. aureus, with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis.
Assuntos
COVID-19 , Interferon Tipo I , Trombose , Humanos , Anticoagulantes , Tromboplastina , Fumarato de Dimetilo/farmacologia , Fumarato de Dimetilo/uso terapêutico , Escherichia coli , Inflamação , Lipopolissacarídeos , Staphylococcus aureus , Trombina , SARS-CoV-2 , Macrófagos , CaspasesRESUMO
Charge transfer (CT) interaction induced formation of a hierarchical supramolecular assembly has attracted attention due to its wide diversity of structural and functional characteristics. In the present work, we report the generation of green luminescent microspheres from the charge transfer interaction induced co-assembly of a bis-naphthyl dipicolinic amide (DPA) derivative with tetracyanobenzene (TCNB) for the first time. The properties of these self-assemblies were studied both in solution and the solid-state using spectroscopic and a variety of microscopy techniques. The X-ray crystal structure analysis showed a mixed stack arrangement of DPA and TCNB. The molecular orbital and energy level calculations confirm the charge transfer complex formation between DPA and TCNB. Furthermore, energy transfer was observed from the green luminescent CT complex to a red-emitting dye, pyronin Y, in the microsphere matrix, leading to the formation of a light-harvesting tri-component self-assembly.
RESUMO
CD44 is emerging as an important receptor biomarker for various cancers. Amongst these is oral cancer, where surgical resection remains an essential mode of treatment. Unfortunately, surgery is frequently associated with permanent disfigurement, malnutrition, and functional comorbidities due to the difficultly of tumour removal. Optical imaging agents that can guide tumour tissue identification represent an attractive approach to minimising the impact of surgery. Here, we report the synthesis of a water-soluble fluorescent probe, namely HA-FA-HEG-OE (compound 1), that comprises components originating from natural sources: oleic acid, ferulic acid and hyaluronic acid. Compound 1 was found to be non-toxic, displayed aggregation induced emission and accumulated intracellularly in vesicles in SCC-9 oral squamous cells. The uptake of 1 was fully reversible over time. Internalization of compound 1 occurs through receptor mediated endocytosis; uniquely mediated through the CD44 receptor. Uptake is related to tumorigenic potential, with non-tumorigenic, dysplastic DOK cells and poorly tumorigenic MCF-7 cells showing only low intracellular levels and highlighting the critical role of endocytosis in cancer progression and metastasis. Together, the recognised importance of CD44 as a cancer stem cell marker in oral cancer, and the reversible, non-toxic nature of 1, makes it a promising agent for real time intraoperative imaging.
Assuntos
Produtos Biológicos , Portadores de Fármacos , Corantes Fluorescentes/administração & dosagem , Imagem Molecular/métodos , Neoplasias Bucais/diagnóstico por imagem , Sobrevivência Celular/efeitos dos fármacos , Ácidos Cumáricos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Ácido Hialurônico/química , Estrutura Molecular , Neoplasias Bucais/metabolismo , Imagem Óptica/métodos , Análise EspectralRESUMO
A family of six Ru(II) polypyridyl complexes (1-6) which contain phenanthroline-based ligands functionalized with alkyl chains of different lengths (one methyl group, 10 and 21 carbon alkyl chains) and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesized and characterized. The influence of the alkyl chain length on their photophysical and photochemical properties as well as in their photobiological applications has been elucidated by monitoring the changes in their MLCT-centered absorption and emission bands. The presence of one methyl group or 10 carbon alkyl chains does not seem to significantly affect the photophysical and photochemical properties of the resulting Ru(II) complexes when compared to the well-known [Ru(phen)3]2+ and [Ru(TAP)2phen]2+. However, an effect on their emission properties and in their ability to photosensitize singlet oxygen is observed for the Ru(II) complexes containing 21 carbon alkyl chains. The binding of these complexes to salmon testes DNA (stDNA) was investigated by observing the changes in the photophysical properties. Complexes 1, 2, 4, and 5 all showed changes in their MLCT bands that could be analyzed using conventional fitting methods, such as the Bard equation. In contrast, complexes 3 and 6, possessing long aliphatic chains, gave rise to nonclassic behavior. In addition to these analyses, both thermal denaturation and circular dichroism studies of 1-6 were carried out in the presence of stDNA which confirmed that these complexes bind to DNA. Confocal microscopy and viability studies in HeLa cervical cancer cells reveal an alkyl chain-length dependence on the cellular uptake and cytotoxicity of the resulting Ru(II) complexes due to an enhancement of their lipophilicity with increasing alkyl chain length. Thus, complexes containing 10 and 21 carbon alkyl chains are rapidly taken up into HeLa cells and, in particular, those with 21 carbon alkyl chains show a significant phototoxicity against the same cell line. Therefore, this study provides further insight into the possible modulation of the photophysical, photochemical, and photobiological properties of Ru(II) polypyridyl complexes by varying the length of the alkyl chains attached to the polypyridyl ligands coordinated to the Ru(II) center and the nature of the auxiliary groups, which we show has a significant effect on photophysical and biological properties.
Assuntos
Complexos de Coordenação , Rutênio , Carbono , Complexos de Coordenação/farmacologia , DNA , Células HeLa , Humanos , Ligantes , Fenantrolinas , Rutênio/farmacologiaRESUMO
Two new dinuclear Ru(ii) polypyridyl complexes containing an alkyl disulphide functionalised bipyridine-based ligand and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesised and characterised. Their attachment onto the surface of gold nanoparticles (AuNPs, average diameter of ca. 2.5 nm) resulted in the formation of two new water-soluble Ru(ii)-AuNP conjugates that combine the advantageous properties of both moieties. Both free complexes show the attractive photophysical properties of Ru(ii) polypyridyl complexes and a rapid cellular uptake in HeLa cervical cancer cells. However, their corresponding gold conjugates displayed lower quantum yields than those determined for the free complexes presumed to be due to an energy transfer quenching of the Ru(ii) luminescence by interaction with the gold surface. Despite their diminished luminescence, confocal fluorescence microscopy studies revealed that the Ru(ii)-AuNP conjugates are successfully internalised into HeLa cells and better tolerated than their free complex counterparts after 24 h incubation, which makes them potential luminescent nanomaterials for bioimaging applications.
Assuntos
Complexos de Coordenação/síntese química , Corantes Fluorescentes/síntese química , Ouro/química , Nanopartículas Metálicas/química , Nanoconjugados/química , Rutênio/química , 2,2'-Dipiridil/síntese química , Permeabilidade da Membrana Celular , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Ligantes , Naftacenos/síntese química , Imagem Óptica , Fenantrolinas/síntese química , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Two water-soluble amphiphilic Ru(ii) polypyridyl complexes containing N-1,10-phenanthrolin-5-yldocosanamide and 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ligands were synthesised and their photophysical and photobiological properties evaluated; both complexes showed a rapid cellular uptake and displayed phototoxicity against HeLa cervical cancer cells.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Tensoativos/farmacologia , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Células HeLa , Humanos , Ligantes , Imagem Óptica , Piridinas/química , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Rutênio/farmacologia , Solubilidade , Tensoativos/química , Água/químicaRESUMO
The photoreceptor outer segment is the canonical example of a modified and highly specialized cilium, with an expanded membrane surface area in the form of disks or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localization of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display significantly reduced shed outer segment material/phagosomes in the RPE at 1 month post fertilization (mpf), but otherwise normal visual function up to 21 dpf and retinal structure up to 12 mpf. Cone photoreceptor-specific transgenic reporter lines show Rab28 localizes almost exclusively to outer segments, independently of GTP/GDP nucleotide binding. Co-immunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, phosphodiesterase 6C and guanylate cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy.
RESUMO
Motile and immotile (or primary) cilia are microtubule-based structures that mediate multiple cellular functions, including the transduction of environmental cues, developmental signaling, cellular motility, and modulation of fluid flow. Although their core architectures are similar, motile and primary cilia exhibit marked structural differences that underlie distinct functional properties. However, the extent to which ciliogenesis mechanisms are shared between these different cilia types is not fully described. Here, we report that the atypical MAP kinase MAPK15 (ERK7/8), implicated in the formation of vertebrate motile cilia, also regulates the formation of primary cilia in Caenorhabditis elegans sensory neurons and human cells. We find that MAPK15 localizes to a basal body subdomain with the ciliopathy protein BBS7 and to cell-cell junctions. MAPK15 also regulates the localization of ciliary proteins involved in cilium structure, transport, and signaling. Our results describe a primary cilia-related role for this poorly studied member of the MAPK family in vivo, and indicate a broad requirement for MAPK15 in the formation of multiple ciliary classes across species.
Assuntos
Caenorhabditis elegans/genética , Cílios/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Células Receptoras Sensoriais/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Movimento Celular/genética , Humanos , Microtúbulos , Transporte Proteico/genética , Transdução de SinaisRESUMO
The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.
Assuntos
Cerebelo/anormalidades , Cílios/metabolismo , Proteínas de Membrana/metabolismo , Retina/anormalidades , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cerebelo/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Proteínas de Membrana/genética , Retina/metabolismoRESUMO
The passive micro-structural mechanical response of muscle tissue is important for numerous medical applications. However, the recently observed tension/compression asymmetry in porcine muscle remains poorly explained. In particular there remains a lack of understanding of how external tension or compression applied in the fibre or cross-fibre direction translates internally to deformation of muscle fibres and the extra-cellular matrix. Accordingly, fresh porcine skeletal muscle tissue was harvested, deformed by 30% in uniaxial tension or compression in both the fibre and cross-fibre directions and prepared for optical microscope, polarised light microscope and SEM analysis. The average deformed specimen results were compared to the average control results in each case. For compressive or tensile stretch applied in the muscle fibre direction the average measured muscle fibre cross-sectional area changes are in close correspondence with predictions based on global Poisson's ratio measurements and these deformation modes did not cause shape changes in the muscle fibre cross-sections. However, muscle tissue reacted to the applied cross-fibre direction deformations as follows: compression flattened muscle fibre cross-sections, aligning them perpendicular to the direction of the applied deformation while tensile deformations stretched the cross-sections of muscle fibres, aligning them parallel to the direction of applied deformation. No evidence of structural reorganisation of endomysium collagen fibres in response to applied stretch was observed. The observed micro-structural responses do not appear to be influenced by the surrounding endomysium, but appear to be significantly influenced by proximity to the perimysium network. It is hypothesised that the perimysium and its interaction with the surrounding muscle fibres is therefore likely to be predominantly responsible for the tension/compression asymmetry observed in macroscopic tests of passive skeletal muscle stress strain behaviour.
Assuntos
Fenômenos Mecânicos , Músculo Esquelético , Animais , Fenômenos Biomecânicos , Força Compressiva , Feminino , Luz , Microscopia Eletrônica de Varredura , Músculo Esquelético/ultraestrutura , Fenômenos Ópticos , SuínosRESUMO
Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14, 47 and 169. We have constructed the single, double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the 'fast' thiol (pK 7.4), and define C169 as the alkaline reactive residue with a pK(a) of 8.6. Only a small proportion of C14 is reactive with 5,5'-dithiobis-(2-nitrobenoic acid) (DTNB) at pH 9 in the C47A/C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics, but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited 'ping pong' kinetics, consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione (GSH) complex to form an enzyme-GS(-) (unprotonated) complex is essentially irreversible. Intriguingly, similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is 'mirrored' in the crystal structure of the C47A mutant, which binds the p-nitrobenzyl moiety of p-nitrobenzyglutathione in distinct orientations in the two crystallographic subunits. The asymmetry seen in this structure for product binding is associated with absence of a water molecule W0 in the standard wild-type conformation of product binding that is clearly identifiable in the new structure, which may represent a structural model for binding of incoming GSH prior to displacement of W0. Elimination of W0 as a hydroxonium ion may be the mechanism for the initial proton extrusion from the active site.