RESUMO
Machine learning methods hold the promise to reduce the costs and the failure rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases, where the development of disease-modifying drugs has been particularly challenging. To address this problem, we describe here a machine learning approach to identify small molecule inhibitors of α-synuclein aggregation, a process implicated in Parkinson's disease and other synucleinopathies. Because the proliferation of α-synuclein aggregates takes place through autocatalytic secondary nucleation, we aim to identify compounds that bind the catalytic sites on the surface of the aggregates. To achieve this goal, we use structure-based machine learning in an iterative manner to first identify and then progressively optimize secondary nucleation inhibitors. Our results demonstrate that this approach leads to the facile identification of compounds two orders of magnitude more potent than previously reported ones.
Assuntos
Descoberta de Drogas , Aprendizado de Máquina , Agregados Proteicos , alfa-Sinucleína , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , Humanos , Descoberta de Drogas/métodos , Agregados Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Relação Estrutura-AtividadeRESUMO
Lewy bodies (LB) are aberrant protein accumulations observed in the brain cells of individuals affected by Parkinson's disease (PD). A comprehensive analysis of LB proteome identified over a hundred proteins, many co-enriched with α-synuclein, a major constituent of LB. Within this context, OTUB1, a deubiquitinase detected in LB, exhibits amyloidogenic properties, yet the mechanisms underlying its aggregation remain elusive. In this study, we identify two critical sites in OTUB1-namely, positions 133 and 173-that significantly impact its amyloid aggregation. Substituting alanine at position 133 and lysine at position 173 enhances both thermodynamic and kinetic stability, effectively preventing amyloid aggregation. Remarkably, lysine at position 173 demonstrates the highest stability without compromising enzymatic activity. The increased stability and inhibition of amyloid aggregation are attributed mainly to the changes in the specific microenvironment at the hotspot. In our exploration of the in-vivo co-occurrence of α-synuclein and OTUB1 in LB, we observed a synergistic modulation of each other's aggregation. Collectively, our study unveils the molecular determinants influencing OTUB1 aggregation, shedding light on the role of specific residues in modulating aggregation kinetics and structural transition. These findings contribute valuable insights into the complex interplay of amino acid properties and protein aggregation, with potential implications for understanding broader aspects of protein folding and aggregation phenomena.
Assuntos
alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/química , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/química , Agregados Proteicos , Corpos de Lewy/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Amiloide/metabolismo , Amiloide/química , Estabilidade Proteica , Estabilidade Enzimática , CinéticaRESUMO
Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular ß-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register ß-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.
Assuntos
Príons , Animais , Camundongos , Microscopia Crioeletrônica , Genótipo , Proteínas Priônicas/genética , Príons/metabolismo , Conformação ProteicaRESUMO
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with average lifespan of 2-5 years after diagnosis. The identification of novel prognostic and pharmacodynamic biomarkers are needed to facilitate therapeutic development. Metalloprotein human superoxide dismutase 1 (SOD1) is known to accumulate and form aggregates in patient neural tissue with familial ALS linked to mutations in their SOD1 gene. Aggregates of SOD1 have also been detected in other forms of ALS, including the sporadic form and the most common familial form linked to abnormal hexanucleotide repeat expansions in the Chromosome 9 open reading frame 72 (C9ORF72) gene. Here, we report the development of a real-time quaking-induced conversion (RT-QuIC) seed amplification assay using a recombinant human SOD1 substrate to measure SOD1 seeding activity in postmortem spinal cord and motor cortex tissue from persons with different ALS etiologies. Our SOD1 RT-QuIC assay detected SOD1 seeds in motor cortex and spinal cord dilutions down to 10-5. Importantly, we detected SOD1 seeding activity in specimens from both sporadic and familial ALS cases, with the latter having mutations in either their SOD1 or C9ORF72 genes. Analyses of RT-QuIC parameters indicated similar lag phases in spinal cords of sporadic and familial ALS patients, but higher ThT fluorescence maxima by SOD1 familial ALS specimens and sporadic ALS thoracic cord specimens. For a subset of sporadic ALS patients, motor cortex and spinal cords were examined, with seeding activity in both anatomical regions. Our results suggest SOD1 seeds are in ALS patient neural tissues not linked to SOD1 mutation, suggesting that SOD1 seeding activity may be a promising biomarker, particularly in sporadic ALS cases for whom genetic testing is uninformative.
Assuntos
Esclerose Lateral Amiotrófica , Biomarcadores , Medula Espinal , Superóxido Dismutase-1 , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Córtex Motor/patologia , Córtex Motor/metabolismo , Mutação/genética , Medula Espinal/patologia , Medula Espinal/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Biomarcadores/análiseRESUMO
Parkinson's disease is a neurodegenerative movement disorder associated with the intracellular aggregation of α-synuclein (α-syn). Cytotoxicity is mainly associated with the oligomeric species (αSOs) formed at early stages in α-syn aggregation. Consequently, there is an intense focus on the discovery of novel inhibitors such as peptides to inhibit oligomer formation and toxicity. Here, using peptide arrays, we identified nine peptides with high specificity and affinity for αSOs. Of these, peptides p194, p235, and p249 diverted α-syn aggregation from fibrils to amorphous aggregates with reduced ß-structures and increased random coil content. However, they did not reduce αSO cytotoxicity and permeabilization of large anionic unilamellar vesicles. In parallel, we identified a non-self-aggregating peptide (p216), derived from the cell-penetrating peptide penetratin, which showed 12-fold higher binding affinity to αSOs than to α-syn monomers (Kdapp 2.7 and 31.2 µM, respectively). p216 reduced αSOs-induced large anionic unilamellar vesicle membrane permeability at 10-1 to 10-3 mg/ml by almost 100%, was not toxic to SH-SY5Y cells, and reduced αSOs cytotoxicity by about 20%. We conclude that p216 is a promising starting point from which to develop peptides targeting toxic αSOs in Parkinson's disease.
Assuntos
Peptídeos Penetradores de Células , Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Peptídeos Penetradores de Células/isolamento & purificação , Peptídeos Penetradores de Células/farmacologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Linhagem Celular TumoralRESUMO
Double-stranded DNA bears the highest linear negative charge density (2e- per base-pair) among all biopolymers, leading to strong interactions with cations and dipolar water, resulting in the formation of a dense 'condensation layer' around DNA. Interactions involving proteins and ligands binding to DNA are primarily governed by strong electrostatic forces. Increased salt concentrations impede such electrostatic interactions - a situation that prevails in oceanic species due to their cytoplasm being enriched with salts. Nevertheless, how these interactions' dynamics are affected in crowded hypersaline environments remains largely unexplored. Here, we employ steady-state and time-resolved fluorescence Stokes shifts (TRFSS) of a DNA-bound ligand (DAPI) to investigate the static and dynamic solvation properties of DNA in the presence of two divalent cations, magnesium (Mg2+), and calcium (Ca2+) at varying high to very-high concentrations of 0.15 M, 1 M and 2 M. We compare the results to those obtained in physiological concentrations (0.15 M) of monovalent Na+ ions. Combining data from fluorescence femtosecond optical gating (FOG) and time-correlated single photon counting (TCSPC) techniques, dynamic fluorescence Stokes shifts in DNA are analysed over a broad range of time-scales, from 100 fs to 10 ns. We find that while divalent cation crowding strongly influences the DNA stability and ligand binding affinity to DNA, the dynamics of DNA solvation remain remarkably similar across a broad range of five decades in time, even in a high-salinity crowded environment with divalent cations, as compared to the physiological concentration of the Na+ ion. Steady-state and time-resolved data of the DNA-groove-bound ligand are seemingly unaffected by ion-crowding in hypersaline solution, possibly due to ions being mostly displaced by the DNA-bound ligand. Furthermore, the dynamic coupling of cations with nearby water may possibly contribute to a net-neutral effect on the overall collective solvation dynamics in DNA, owing to the strong anti-correlation of their electrostatic interaction energy fluctuations. Such dynamic scenarios may persist within the cellular environment of marine life and other biological cells that experience hypersaline conditions.
Assuntos
DNA , Salinidade , Cátions Bivalentes , Ligantes , DNA/química , Íons , Sódio , Água/química , Cátions , Cátions MonovalentesRESUMO
Amyloid diseases are of major concern all over the world due to a number of factors including: (i) aging population, (ii) increasing life span and (iii) lack of effective pharmacotherapy options. The past decade has seen intense research in discovering disease-modifying multi-targeting small molecules as therapeutic options. In recent years, targeting the amyloid cascade has emerged as an attractive strategy to discover novel neurotherapeutics. Formation of amyloid species, with different degrees of solubility and neurotoxicity is associated with the gradual decline in cognition leading to dementia/cell dysfunction. Here, in this chapter, we have described the recent scenario of amyloid diseases with a great deal of information about the structural features of oligomers, protofibrils and fibrils. Also, comprehensive details have been provided to differentiate the degree of toxicity associated with prefibrillar aggregates. Moreover, a review of the technologies that aid characterisation of oligomer, protofibrils and fibrils as well as various inhibition strategies to overcome protein fibrillation are also discussed.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Amiloidose , Amiloide/metabolismo , Amiloidose/metabolismo , Amiloidose/patologia , Humanos , Agregação Patológica de Proteínas/patologiaRESUMO
Amyloid fibrillation is associated with several human maladies, such as Alzheimer's, Parkinson's, Huntington's diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti-inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in a linear fashion by influencing the nucleation phase as assayed by thioflavin T fluorescence, transmission electron microscopy, and dynamic light scattering. Furthermore, circular dichroism analysis supported the DSC results, showing that IBFN binds to the native state of proteins and almost completely prevents their tendency to lose secondary and tertiary structures. Cell toxicity assay confirms that species formed in the presence of IBFN are less toxic to neuronal cells (SH-SY5Y). These results demonstrate the feasibility of using a small molecule to stabilize the native state of proteins, thereby preventing the amyloidogenic conformational changes, which appear to be the common link in several human amyloid diseases.
RESUMO
This review article provides an overview of the different species that α-synuclein aggregates can populate. It also attempts to reconcile conflicting views regarding the cytotoxic roles of oligomers versus fibrils. α-synuclein, while highly dynamic in the monomeric state, can access a large number of different assembly states. Depending on assembly conditions, these states can interconvert over different timescales. The fibrillar state is the most thermodynamically favored due to the many stabilizing interactions formed between each monomeric unit, but different fibrillar types form at different rates. The end distribution is likely to reflect kinetic partitioning as much as thermodynamic equilibra. In addition, metastable oligomeric species, some of which are on-pathway and others off-pathway, can be populated for remarkably long periods of time. Chemical modifications (phosphorylation, oxidation, covalent links to ligands, etc.) perturb these physical interconversions and invariably destabilize the fibrillar state, leading to small prefibrillar assemblies which can coalesce into amorphous states. Both oligomeric and fibrillar species have been shown to be cytotoxic although firm conclusions require very careful evaluation of particle concentrations and is complicated by the great variety and heterogeneity of different experimentally observed states. The mechanistic relationship between oligomers and fibrils remains to be clarified, both in terms of assembly of oligomers into fibrils and potential dissolution of fibrils into oligomers. While oligomers are possibly implicated in the collapse of neuronal homeostasis, the fibrillar state(s) appears to be the most efficient at propagating itself both in vitro and in vivo, pointing to critical roles for multiple different aggregate species in the progression of Parkinson's disease (https://onlinelibrary.wiley.com/page/journal/14714159/homepage/virtual_issues.htm). This article is part of the Special Issue "Synuclein".
Assuntos
Sinucleinopatias/metabolismo , alfa-Sinucleína/química , Amiloide/química , Humanos , Cinética , Corpos de Lewy/química , Peroxidação de Lipídeos , Metais/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Mutação Puntual , Agregação Patológica de Proteínas , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Solubilidade , Relação Estrutura-Atividade , Sinucleinopatias/genética , Termodinâmica , alfa-Sinucleína/genéticaRESUMO
Protein misfolding and aggregation lead to amyloid generation that in turn may induce cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat amyloidogenesis, large number of studies has been paying attention on breakthrough of amyloid inhibitors. In the present work, we aim to access the effect of two drugs, that is, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using various biophysical, imaging, cell viability assay, and computational approaches. We established that both drugs reduce the turbidity, light scattering and fluorescence intensity of amyloid indicator dye thioflavin T. Premixing of drugs with insulin inhibited the nucleation phase and inhibitory potential was boosted by increasing the concentration of the drug. Moreover, addition of drugs at the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid exhibited enhanced inhibitory effects on insulin fibrillation in comparison to acetyl group. It may be due to presence of amino group that helps it to prolong the nucleation phase with strong binding as well as disruption of aromatic and hydrophobic stacking that plays a key role in amyloid progression.
Assuntos
Amiloide , Insulina , Mesalamina/química , Ácido Salicílico/química , Amiloide/química , Amiloide/farmacologia , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Insulina/química , Insulina/farmacologiaRESUMO
Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer's and Parkinson's diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha helical structure of protein during fibrillation, reduces the formation of beta sheet rich structure. Furthermore, Dynamic Light Scattering (DLS) and Transmission Electronic Microscopy (TEM) analysis confirmed that CN treated samples exhibited different size distribution and morphology, respectively. In addition, molecular docking results revealed that CN interacts with insulin through hydrophobic interactions as well as hydrogen bonding, and the Hemolytic assay confirmed the non-hemolytic activity of CN on human RBCs. For future research, this study may assist in the rational designing of molecules against amyloid formation.
Assuntos
Amiloide/química , Capreomicina/química , Insulina/química , Simulação de Acoplamento Molecular , Agregados Proteicos , Amiloide/ultraestrutura , Animais , Capreomicina/farmacologia , Bovinos , HumanosRESUMO
Protein aggregation into oligomers and fibrils are associated with many human pathophysiologies. Compounds that modulate protein aggregation and interact with preformed fibrils and convert them to less toxic species, expect to serve as promising drug candidates and aid to the drug development efforts against aggregation diseases. In present study, the kinetics of amyloid fibril formation by human insulin (HI) and the anti-amyloidogenic activity of ascorbic acid (AA) were investigated by employing various spectroscopic, imaging and computational approaches. We demonstrate that ascorbic acid significantly inhibits the fibrillation of HI in a dose-dependent manner. Interestingly ascorbic acid destabilise the preformed amyloid fibrils and protects human neuroblastoma cell line (SH- SY5Y) against amyloid induced cytotoxicity. The present data signifies the role of ascorbic acid that can serve as potential molecule in preventing human insulin aggregation and associated pathophysiologies.
Assuntos
Amiloide/síntese química , Ácido Ascórbico/química , Insulina/química , Insulina/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Ácido Ascórbico/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Neurônios/efeitos dos fármacosRESUMO
As a part of our continuing program on the synthesis of steroidal heterocycles, it has been prepared a series of novel steroidal pyrimidine derivatives 4-6via TMSCl, steroidal ketones (1c-3c), urea and benzaldehyde. The systems presented here, are novel scaffolds and have not been described before at 6th position of steroidal-6-one (1c-3c). Structural assignment of newly synthesized compounds was performed by DFT/B3LYP calculations as well as spectral and analytical data. The interactions of compounds (4-6) with HSA were studied by fluorescence spectroscopy, DLS, CD and molecular docking, under imitated physiological conditions. The antitumor activity has been tested in vitro against three cancer cell lines MDA-MB231 (breast carcinoma), HeLa (human cervical carcinoma), HepG2 (hepatic carcinoma) and one non-cancer normal cell lines, PBMCs (peripheral blood mononuclear cell) by MTT assay. In addition, in vitro antioxidant activity and apoptosis assay of the synthesized compounds (4-6) have also been investigated.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Simulação de Acoplamento Molecular , Pirimidinas/farmacologia , Teoria Quântica , Esteroides/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antioxidantes/síntese química , Antioxidantes/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Esteroides/síntese química , Esteroides/química , Relação Estrutura-AtividadeRESUMO
Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of keratins on ß-cell organisation and systemic glucose control was assessed using keratin 8 (K8) wild-type (K8(+/+)) and K8 knockout (K8(-/-)) mice. Islet ß-cell keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage was assessed in K8(+/+) and K8(-/-) mice. K8 and K18 were the predominant keratins in islet ß-cells and K8(-/-) mice expressed only remnant K18 and K7. K8 deletion resulted in lower fasting glucose levels, increased glucose tolerance and insulin sensitivity, reduced glucose-stimulated insulin secretion and decreased pancreatic insulin content. GLUT2 localisation and insulin vesicle morphology were disrupted in K8(-/-) ß-cells. The increased levels of cytoplasmic GLUT2 correlated with resistance to high-dose STZ-induced injury in K8(-/-) mice. However, K8 deletion conferred no long-term protection from STZ-induced diabetes and prolonged STZ-induced stress caused increased exocrine damage in K8(-/-) mice. ß-cell keratin upregulation occurred 2 weeks after treatments with low-dose STZ in K8(+/+) mice and in diabetic NOD mice, suggesting a role for keratins, particularly in non-acute islet stress responses. These results demonstrate previously unrecognised functions for keratins in ß-cell intracellular organisation, as well as for systemic blood glucose control under basal conditions and in diabetes-induced stress.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Células Secretoras de Insulina/metabolismo , Queratina-8/fisiologia , Estresse Fisiológico , Animais , Glicemia , Diabetes Mellitus Experimental/patologia , Feminino , Transportador de Glucose Tipo 2/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Queratina-18/metabolismo , Queratina-7/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/patologiaRESUMO
This paper concerns the tear properties and behavior of Bombyx mori (B. Mori) silk cocoons. The tear resistance of cocoon layers is found to increase progressively from the innermost layer to the outermost layer. Importantly, the increase in tear strength correlates with increased porosity, which itself affects fiber mobility. We propose a microstructural mechanism for tear failure, which begins with fiber stretching and sliding, leading to fiber piling, and eventuating in fiber fracture. The direction of fracture is then deemed to be a function of the orientation of piled fibers, which is influenced by the presence of junctions where fibers cross at different angles and which may then act as nucleating sites for fiber piling. The interfaces between cocoon wall layers in B. mori cocoon walls account for 38% of the total wall tear strength. When comparing the tear energies and densities of B. mori cocoon walls against other materials, we find that the B. mori cocoon walls exhibit a balanced trade-off between tear resistance and lightweightness.
RESUMO
Guanine-rich single-stranded DNA folds into G-quadruplex DNA (GqDNA) structures, which play crucial roles in various biological processes. These structures are also promising targets for ligands, potentially inducing antitumor effects. While thermodynamic parameters of ligand/DNA interactions are well-studied, the kinetics of ligand interaction with GqDNA, particularly in cell-like crowded environments, remain less explored. In this study, we investigate the impact of molecular crowding agents (glucose, sucrose, and ficoll 70) at physiologically relevant concentrations (20% w/v) on the association and dissociation rates of the benzophenoxazine-core based ligand, cresyl violet (CV), with human telomeric antiparallel-GqDNA. We utilized fluorescence correlation spectroscopy (FCS) along with other techniques. Our findings reveal that crowding agents decrease the binding affinity of CV to GqDNA, with the most significant effect-a nearly three-fold decrease-observed with ficoll 70. FCS measurements indicate that this decrease is primarily due to a viscosity-induced slowdown of ligand association in the crowded environment. Interestingly, dissociation rates remain largely unaffected by smaller crowders, with only small effect observed in presence of ficoll 70 due to direct but weak interaction between the ligand and ficoll. These results along with previously reported data provide valuable insights into ligand/GqDNA interactions in cellular contexts, suggesting a conserved mechanism of saccharide crowder influence, regardless of variations in GqDNA structure and ligand binding mode. This underscores the importance of considering crowding effects in the design and development of GqDNA-targeted drugs for potential cancer treatment.
Assuntos
Quadruplex G , Espectrometria de Fluorescência , Espectrometria de Fluorescência/métodos , Ligantes , Cinética , Humanos , DNA/químicaRESUMO
Ras signaling and glycosylphosphatidylinositol (GPI) biosynthesis are mutually inhibitory in S. cerevisiae (Sc). The inhibition is mediated via an interaction of yeast Ras2 with the Eri1 subunit of its GPI-N-acetylglucosaminyl transferase (GPI-GnT), the enzyme catalyzing the very first GPI biosynthetic step. In contrast, Ras signaling and GPI biosynthesis in C. albicans (Ca) are mutually activated and together control the virulence traits of the human fungal pathogen. What might be the role of Eri1 in this pathogen? The present manuscript addresses this question while simultaneously characterizing the cellular role of CaEri1. It is either nonessential or required at very low levels for cell viability in C. albicans. Severe depletion of CaEri1 results in reduced GPI biosynthesis and cell wall defects. It also produces hyperfilamentation phenotypes in Spider medium as well as in bicarbonate medium containing 5% CO2, suggesting that both the Ras-dependent and Ras-independent cAMP-PKA pathways for hyphal morphogenesis are activated in these cells. Pull-down and acceptor-photobleaching FRET experiments suggest that CaEri1 does not directly interact with CaRas1 but does so through CaGpi2, another GPI-GnT subunit. We showed previously that CaGpi2 is downstream of CaEri1 in cross talk with CaRas1 and for Ras-dependent hyphal morphogenesis. Here we show that CaEri1 is downstream of all GPI-GnT subunits in inhibiting Ras-independent filamentation. CaERI1 also participates in intersubunit transcriptional cross talk within the GPI-GnT, a feature unique to C. albicans. Virulence studies using G. mellonella larvae show that a heterozygous strain of CaERI1 is better cleared by the host and is attenuated in virulence.
RESUMO
Accurate detection of bacterial antibiotic sensitivity is crucial for theranostics and the containment of antibiotic-resistant infections. However, the intricate task of detecting and quantifying the antibiotic-induced changes in the bacterial cytoplasmic membrane, and their correlation with other metabolic pathways leading to antibiotic resistance, poses significant challenges. Using a novel class of 4-aminophthalimide (4AP)-based fluorescent dyes with precisely tailored alkyl chains, namely 4AP-C9 and 4AP-C13, we quantify stress-mediated alterations in E. coli membranes. Leveraging the unique depth-dependent positioning and environment-sensitive fluorescence properties of these dyes, we detect antibiotic-induced membrane damage through single-cell imaging and monitoring the fluorescence peak maxima difference ratio (PMDR) of the dyes within the bacterial membrane, complemented by other methods. The correlation between the ROS-induced cytoplasmic membrane damage and the PMDR of dyes quantifies sensitivity against bactericidal antibiotics, which correlates to antibiotic-induced lipid peroxidation. Significantly, our findings largely extend to clinical isolates of E. coli and other ESKAPE pathogens like K. pneumoniae and Enterobacter subspecies. Our data reveal that 4AP-Cn probes can potentially act as precise scales to detect antibiotic-induced membrane damage ("thinning") occurring at a subnanometer scale through the quantification of dyes' PMDR, making them promising membrane dyes for rapid detection of bacterial antibiotic resistance, distinguishing sensitive and resistant infections with high specificity in a clinical setup.
Assuntos
Antibacterianos , Membrana Celular , Escherichia coli , Corantes Fluorescentes , Testes de Sensibilidade Microbiana , Corantes Fluorescentes/química , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Enterobacter/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacosRESUMO
The appreciation of how conventional and fossil-based materials could be harmful to our planet is growing, especially when considering single-use and non-biodegradable plastics manufactured from fossil fuels. Accordingly, tackling climate change and plastic waste pollution entails a more responsible approach to sourcing raw materials and the adoption of less destructive end-of-life pathways. Livestock animals, in particular ruminants, process plant matter using a suite of mechanical, chemical and biological mechanisms through the act of digestion. The manure from these "living bioreactors" is ubiquitous and offers a largely untapped source of lignocellulosic biomass for the development of bio-based and biodegradable materials. In this review, we assess recent studies made into manure-based cellulose materials in terms of their material characteristics and implications for sustainability. Despite the surprisingly diverse body of research, it is apparent that progress towards the commercialisation of manure-derived cellulose materials is hindered by a lack of truly sustainable options and robust data to assess the performance against conventional materials alternatives. Nanocellulose, a natural biopolymer, has been successfully produced by living bioreactors and is presented as a candidate for future developments. Life cycle assessments from non-wood sources are however minimal, but there are some initial indications that manure-derived nanocellulose would offer environmental benefits over traditional wood-derived sources.
Assuntos
Celulose , Esterco , Animais , Poluição Ambiental , Plásticos , Reatores BiológicosRESUMO
Small soluble oligomers of the protein α-synuclein (αSO) have been linked to disruptions in neuronal homeostasis, contributing to the development of Parkinson's Disease (PD). While this makes αSO an obvious drug target, the development of effective therapeutics against αSO is challenged by its low abundance and structural and morphological complexity. Here, we employ two different approaches to neutralize toxic interactions made by αSOs with different cellular components. First, we use available data to identify four neuronal proteins as likely candidates for αSO interactions, namely Cfl1, Uchl1, Sirt2 and SerRS. However, despite promising results when immobilized, all 4 proteins only bind weakly to αSO in solution in microfluidic assays, making them inappropriate for screening. In contrast, the formation of stable contacts formed between αSO and vesicles consisting of anionic lipids not only mimics a likely biological role of αSO but also provided a platform to screen two small molecule libraries for disruptors of these contacts. Of the 7 best leads obtained in this way, 2 significantly impaired αSO contacts with other proteins in a sandwich ELISA assay using αSO-binding monoclonal antibodies and nanobodies. In addition, 5 of these leads suppressed α-synuclein amyloid formation. Thus, a repurposing screening that directly targets a key culprit in PD pathogenesis shows therapeutic potential.