RESUMO
Nonlinear biomolecular interactions on membranes drive membrane remodeling crucial for biological processes including chemotaxis, cytokinesis, and endocytosis. The complexity of biomolecular interactions, their redundancy, and the importance of spatiotemporal context in membrane organization impede understanding of the physical principles governing membrane mechanics. Developing a minimal in vitro system that mimics molecular signaling and membrane remodeling while maintaining physiological fidelity poses a major challenge. Inspired by chemotaxis, we reconstructed chemically regulated actin polymerization inside vesicles, guiding membrane self-organization. An external, undirected chemical input induced directed actin polymerization and membrane deformation uncorrelated with upstream biochemical cues, suggesting symmetry breaking. A biophysical model incorporating actin dynamics and membrane mechanics proposes that uneven actin distributions cause nonlinear membrane deformations, consistent with experimental findings. This protocellular system illuminates the interplay between actin dynamics and membrane shape during symmetry breaking, offering insights into chemotaxis and other cell biological processes.
Assuntos
Actinas , Células Artificiais , Membrana Celular , Polimerização , Actinas/metabolismo , Células Artificiais/metabolismo , Células Artificiais/química , Membrana Celular/metabolismo , Quimiotaxia , Modelos BiológicosRESUMO
Combination of waning immunity and lower effectiveness against new SARS-CoV-2 variants of approved COVID-19 vaccines necessitates new vaccines. We evaluated two doses, 28 days apart, of ARCT-154, a self-amplifying mRNA COVID-19 vaccine, compared with saline placebo in an integrated phase 1/2/3a/3b controlled, observer-blind trial in Vietnamese adults (ClinicalTrial.gov identifier: NCT05012943). Primary safety and reactogenicity outcomes were unsolicited adverse events (AE) 28 days after each dose, solicited local and systemic AE 7 days after each dose, and serious AEs throughout the study. Primary immunogenicity outcome was the immune response as neutralizing antibodies 28 days after the second dose. Efficacy against COVID-19 was assessed as primary and secondary outcomes in phase 3b. ARCT-154 was well tolerated with generally mild-moderate transient AEs. Four weeks after the second dose 94.1% (95% CI: 92.1-95.8) of vaccinees seroconverted for neutralizing antibodies, with a geometric mean-fold rise from baseline of 14.5 (95% CI: 13.6-15.5). Of 640 cases of confirmed COVID-19 eligible for efficacy analysis most were due to the Delta (B.1.617.2) variant. Efficacy of ARCT-154 was 56.6% (95% CI: 48.7- 63.3) against any COVID-19, and 95.3% (80.5-98.9) against severe COVID-19. ARCT-154 vaccination is well tolerated, immunogenic and efficacious, particularly against severe COVID-19 disease.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Humanos , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , COVID-19/imunologia , Feminino , Masculino , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/imunologia , Pessoa de Meia-Idade , Imunogenicidade da Vacina , Adulto Jovem , Eficácia de Vacinas , Vietnã , Adolescente , Vacinas de mRNA , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/administração & dosagemRESUMO
The Bin-Amphiphysin-Rvs (BAR) domain of endophilin binds to the cell membrane and shapes it into a tubular shape for endocytosis. Endophilin has a Src-homology 3 (SH3) domain at their C-terminal. The SH3 domain interacts with the proline-rich motif (PRM) that is found in proteins such as neural Wiskott-Aldrich syndrome protein (N-WASP). Here, we re-examined the binding sites of the SH3 domain of endophilin in N-WASP by machine learning-based prediction and identified the previously unrecognized binding site. In addition to the well-recognized PRM at the central proline-rich region, we found a PRM in front of the N-terminal WASP homology 1 (WH1) domain of N-WASP (NtPRM) as a binding site of the endophilin SH3 domain. Furthermore, the diameter of the membrane tubules in the presence of NtPRM mutant was narrower and wider than that in the presence of N-WASP and in its absence, respectively. Importantly, the NtPRM of N-WASP was involved in the membrane localization of endophilin A2 in cells. Therefore, the NtPRM contributes to the binding of endophilin to N-WASP in membrane remodeling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Proteínas de Transporte/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Domínios de Homologia de src , Fatores de Transcrição/metabolismo , Prolina/metabolismo , Ligação ProteicaRESUMO
The higher-order assembly of Bin-amphiphysin-Rvs (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, into lattice on the membrane is essential for the formation of subcellular structures. However, the regulation of their ordered assembly has not been elucidated. Here, we show that the higher ordered assembly of growth-arrested specific 7 (GAS7), an F-BAR domain protein, is regulated by the multivalent scaffold proteins of Wiskott-Aldrich syndrome protein (WASP)/neural WASP, that commonly binds to the BAR domain superfamily proteins, together with WISH, Nck, the activated small guanosine triphosphatase Cdc42, and a membrane-anchored phagocytic receptor. The assembly kinetics by fluorescence resonance energy transfer monitoring indicated that the GAS7 assembly on liposomes started within seconds and was further increased by the presence of these proteins. The regulated GAS7 assembly was abolished by Wiskott-Aldrich syndrome mutations both in vitro and in cellular phagocytosis. Therefore, Cdc42 and the scaffold proteins that commonly bind to the BAR domain superfamily proteins promoted GAS7 assembly.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteína da Síndrome de Wiskott-Aldrich , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Actinas/metabolismoRESUMO
Fes/Cip4 homology Bin/amphiphysin/Rvs (F-BAR) domains, like all BAR domains, are dimeric units that oligomerize and bind membranes. F-BAR domains are generally coupled to additional domains that function in protein binding or have enzymatic activity. Because of their crescent shape and ability to oligomerize, F-BAR domains have been traditionally viewed as membrane-deformation modules. However, multiple independent studies have provided no evidence that certain F-BAR domains are able to tubulate membrane. Instead, a growing body of literature featuring structural, biochemical, biophysical, and microscopy-based studies supports the idea that the F-BAR domain family can be unified only by their ability to form oligomeric assemblies on membranes to provide platforms for molecular assembly.
Assuntos
Membrana Celular , Membrana Celular/metabolismo , Humanos , Membranas , Ligação ProteicaRESUMO
Coastal urbanization causes environmental modifications that directly and indirectly influence the distribution and functioning of coral reefs. However, the capacity of urban infrastructure to support corals and vertically accrete is less understood. Here, we investigated if coral communities on reefs and seawalls in Singapore are distinct, and examined the environmental variables influencing coral carbonate production. Surveys at 22 sites yielded 134 coral species, with richness significantly higher on reefs. Coral cover and Shannon index did not differ between habitat types. Community composition was distinct between habitat types, with seawalls supporting a higher proportion of massive and thick-plating species. 'Distance from mainland' was the single most important variable influencing normalized carbonate production rates (a function of species-specific linear extension rate and skeletal bulk density and site coral cover), which were higher further from the mainland where human activity and development pressures were greater. Our results indicate that environmental filtering strongly shapes coral communities and may influence ecosystem functioning in Singapore's urbanized reef system. The findings will guide the management of reefs on increasingly urbanized coastlines.
Assuntos
Antozoários , Animais , Carbonatos , Recifes de Corais , Ecossistema , Humanos , SingapuraRESUMO
Phagocytosis is a cellular process for internalization of micron-sized large particles including pathogens. The Bin-Amphiphysin-Rvs167 (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, impose specific morphologies on lipid membranes. Most BAR domain proteins are thought to form membrane invaginations or protrusions by assembling into helical submicron-diameter filaments, such as on clathrin-coated pits, caveolae, and filopodia. However, the mechanism by which BAR domain proteins assemble into micron-scale phagocytic cups was unclear. Here, we show that the two-dimensional sheet-like assembly of Growth Arrest-Specific 7 (GAS7) plays a critical role in phagocytic cup formation in macrophages. GAS7 has the F-BAR domain that possesses unique hydrophilic loops for two-dimensional sheet formation on flat membranes. Super-resolution microscopy reveals the similar assemblies of GAS7 on phagocytic cups and liposomes. The mutations of the loops abolishes both the membrane localization of GAS7 and phagocytosis. Thus, the sheet-like assembly of GAS7 plays a significant role in phagocytosis.
Assuntos
Macrófagos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fagocitose , Sequência de Aminoácidos , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células HeLa , Humanos , Lipídeos de Membrana/química , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células RAW 264.7 , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Beta-galactosidase (EC 3.2.1.23), a commercially important enzyme, catalyses the hydrolysis of ß-1,3- and ß-1,4-galactosyl bonds of polymer or oligosaccharidesas well as transglycosylation of ß-galactopyranosides. Due to catalytic properties; ß-galactosidase might be useful in the milk industry to hydrolyze lactose and produce prebiotic GOS. The purpose of this study is to characterize ß-galactosidase mutants from B. subtilis. RESULTS: Using error prone rolling circle amplification (epRCA) to characterize some random mutants of the ß-galactosidase (LacA) from B. subtilisVTCC-DVN-12-01, amino acid A301 and F361 has been demonstrated significantly effect on hydrolysis activity of LacA. Mutants A301V and F361Y had markedly reduced hydrolysis activity to 23.69 and 43.22 %, respectively. Mutants the site-saturation of A301 reduced catalysis efficiency of LacA to 20-50 %, while the substitution of F361 by difference amino acids (except tyrosine) lost all of enzymatic activity, indicating that A301 and F361 are important for the catalytic function. Interestingly, the mutant F361Y exhibited enhanced significantly thermostability of enzyme at 45-50 °C. At 45 °C, LacA-361Y retained over 93 % of its original activity for 48 h of incubation, whereas LacA-WT and LacA-301Vwere lost completely after 12 and 24 h of incubation, respectively. The half-life times of LacA-361Y and LacA-301 V were about 26.8 and 2.4 times higher, respectively, in comparison to the half-life time of LacA-WT. At temperature optimum 50 °C, LacA-361Y shows more stable than LacA-WT and LacA-301 V, retaining 79.88 % of its original activities after 2 h of incubation, while the LacA-WT and LacA-301 V lost all essential activities. The half-life time of LacA-361Y was higher 12.7 and 9.39 times than that of LacA-WT and LacA-301 V, respectively. LacA-WT and mutant enzymes were stability at pH 5-9, retained over 90 % activity for 72 h of incubation at 30 °C. However, LacA-WT showed a little bit more stability than LacA-301 V and LacA-361Y at pH 4. CONCLUSIONS: Our findings demonstrated that the amino acids A301V and F361 play important role in hydrolysis activity of ß -galactosidase from B. subtilis. Specially, amino acid F361 had noteworthy effect on both catalytic and thermostability of LacA enzyme, suggesting that F361 is responsible for functional requirement of the GH42 family.