Dual sites for SEC11 on the SNARE SYP121 implicate a binding exchange during secretory traffic

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion. Much of vesicle traffic at the Arabidopsis (Arabidopsis thaliana) plasma membrane is subject to the Sec1/Munc18 protein SEC11, which, along with plasma membrane K+ channels, selectively binds with the SNARE SYP121 to regulate its assembly in complex. How SEC11 binding is coordinated with the K+ channels is poorly understood, as both SEC11 and the channels are thought to compete for the same SNARE binding site. Here, we identify a second binding motif within the N terminus of SYP121 and demonstrate that this motif affects SEC11 binding independently of the F9xRF motif that is shared with the K+ channels. This second, previously unrecognized motif is centered on residues R20R21 of SYP121 and is essential for SEC11 interaction with SYP121. Mutation of the R20R21 motif blocked vesicle traffic without uncoupling the effects of SYP121 on solute and K+ uptake associated with the F9xRF motif; the mutation also mimicked the effects on traffic block observed on coexpression of the dominant-negative SEC11Δ149 fragment. We conclude that the R20R21 motif represents a secondary site of interaction for the Sec1/Munc18 protein during the transition of SYP121 from the occluded to the open conformation that leads to SNARE complex assembly

Membrane voltage as a dynamic platform for spatio-temporal signalling, physiological and developmental regulation

Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic ′currency′ of the membrane. The dynamics of membrane voltage - so-called action, systemic, and variation potentials - have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport - an electrical ′substrate′ - and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signalling via second messengers that rely on ion flux, including cytosolic free Ca2+, H+, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory

Synergy Among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion during Vegetative Growth

Vesicle exocytosis underpins signaling and development in plants and is vital for wall remodeling during cell expansion. Vesicle tethering and fusion are thought to occur sequentially, tethering mediated by the exocyst and fusion driven by assembly of SNARE protein complexes. Interactions between these two protein complexes are known, although insights into their functional consequences are largely unexplored. We now identify a clear hierarchy of interactions leading to secretion in Arabidopsis. Mating-based split-ubiquitin screens and in vivo FRET analyses showed that exocyst EXO70 subunits bind preferentially with cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distributions and suppressed vesicle traffic like the dominant-negative SYP121deltaC, consistent with the epistasis of exo70A1 over the exo70A1syp121 double mutant. However, the exo70A1vamp721 mutant showed a strong synergistic suppression of growth. These data are best explained by a spatiotemporal hierarchy of exocyst recruitment with the R-SNARE to the plasma membrane, plausibly with the VAMP721 longin domain as a nexus for binding

Parent training for disruptive behavior symptoms in attention deficit hyperactivity disorder: a randomized clinical trial

BackgroundAttention-Deficit/Hyperactivity Disorder (ADHD) affects 5% of children and 2.5% of adults worldwide. Comorbidities are frequent, and Oppositional Defiant Disorder (ODD) reaches 50%. Family environment is crucial for the severity of behaviors and for prognosis. In middle-income countries, access to treatment is challenging, with more untreated children than those under treatment. Face-to-face behavioral parent training (PT) is a well-established intervention to improve child behavior and parenting.MethodA clinical trial was designed to compare PT-online and face-to-face effects to a waiting list group. Outcomes were the ADHD and ODD symptoms, parental stress and styles, and quality of life. Families were allocated into three groups: standard treatment (ST), ST + PT online, and ST + Face-to-Face PT. We used repeated measures ANOVA for pre × post treatment analysis corrected for multiple comparisons.Results and discussionParent training was effective in reducing symptoms of ADHD (p = 0.030) and ODD (p = 0.026) irrespective of modality (p = 1.000). The combination of ST and PT was also associated with better quality of life in the physical domain for patients (p = 0.009) and their parents (p = 0.050). In addition to preliminary data, online intervention seems effective for parenting and improving social acceptance of children. The potential to reach many by an online strategy with a self-directed platform may imply effectiveness with a low cost for public health to support parents’ symptoms management

Composição e caracterização química da fração sacarídica do coproduto da biofermentação etanólica da batata-doce (Ipomoea batatas (L.) Lam)

The growing in the demand and need for fuels lead to the search for a renewable and clean source. The biofermentation of sweet potato (Ipomoea batatas (L.) Lam) for the production of biofuel generates a waste which is in the most times disposable in field, but the characteristics of this waste (rich in protein and carbohydrate) makes it susceptible for being studied and other uses exploited, making it a coproduct of the production. The goal of this work was the study of the composition and chemical structure of the carbohydrates found in the coproduct of the fermentation of sweet potato. Underwent this coproduct to two consecutive water extractions, cold-water extraction (CWE) and hot-water extraction (HWE). The extracted, fractioned (oligosaccharides and polysaccharides), hydrolyzed and purified carbohydrates underwent to different chromatography techniques (Thin layer chromatography, high precision liquid chromatography and gas chromatography with mass spectrometry) to clarify the composition and quantity of monosaccharides. Nuclear magnetic resonance (NMR) was used to the structural characterization of the carbohydrates. With the use of high precision techniques (GC-MS and HPLC) it was possible to identify and quantify the sugars. Glucose is the main component founded in the oligosaccharide fraction and galactose in the polysaccharides, was possible to identify the presence of mannose, xylose, rhamnose and arabinose in fewer quantities. The structural analyses show that the polysaccharide fraction has a type I arabinogalactana (composed by (1→4)-β-Galp as the backbone). Those polymers are highly studied because of the benefits when ingested, for example the combat to virus, bifidogenic activity, related to be beneficial to the immune system and to cancer. Therefore, the continued study of this coproduct is necessary and the possible health benefits.A crescente demanda de combustível leva a procura por um que seja produzido a partir de uma fonte limpa e renovável. A biofermentação da batatadoce (Ipomoea batatas (L.) Lam) para a produção de biocombustíveis gera um resíduo que muitas vezes é descartado no campo mas por ser rico em proteínas e carboidratos outros usos e aplicações podem e devem ser exploradas. O objetivo deste trabalho foi estudar a composição e estrutura química fina dos carboidratos presentes no coproduto proveniente da biofermentação etanólica de batata-doce. O coproduto foi submetido a duas extrações aquosas consecutivas, extração aquosa fria (AQF) e uma extração aquosa quente (AQQ). Os carboidratos extraídos, fracionados (oligossacarídeos e polissacarídeos), purificados e hidrolisados foram submetidos a diferentes técnicas cromatográficas (Cromatografia em camada delgada, cromatografia liquida de alta eficiência e cromatografia gasosa acoplada a espectrometria de massas) visando a composição monossacarídica e quantificação destes. A caracterização estrutural dos carboidratos for feita por meio de ressonância magnética nuclear. Foi possível quantificar e identificar por meio de técnicas de alta precisão (CG e CLAE) os monossacarídeos presentes. Glicose é o principal componente da fração oligossacarídica e a galactose da polissacarídica, foi possível ainda identificar a presença de manose, xilose, ramnose e arabinose em menores quantidades. A análise estrutural mostrou que a fração polissacarídica tem como componente uma possível arabinogalactana do tipo I (com cadeia principal composta de (1→4)-β-Galp). Estes polímeros são amplamente estudados em virtude dos seus benéficos quando ingeridos, dentre eles combatem diversos vírus prejudiciais, atividade bifidogênica, benefícios relacionados ao sistema imune e relacionadas ao câncer. Assim, faz-se necessário o estudo continuado deste coproduto além de suas possíveis atividades biológicas

On the mechanisms of receptor-mediated retention of soluble endoplasmic reticulum resident proteins in eukaryotes.

Accumulation of soluble proteins in the endoplasmic reticulum (ER) of plants is mediated by a protein receptor termed ER RETENTION DEFECTIVE 2 (ERD2). To study the mechanism for protein accumulation in the ER, I have optimized a previously established bioassay using Nicotiana benthamiana protoplasts, which proved to be outstanding. The combined use of gain-of- function assays, complementation assays, anti-sense inhibition and confocal laser scanning microscopy allowed me to show that biologically active fluorescent ERD2 fusions are exclusively detected at the Golgi apparatus and do not show ligand-induced redistribution to the ER. I also show that ERD2 dual ER-Golgi distribution is accompanied to lack-of biological function due to the masking a novel C-terminal di-leucine motif. This motif is shown to not promote rapid ER export, but it prevents recycling from the Golgi apparatus back to the ER. Further analysis revealed that the ERD2 C-terminus is necessary but not sufficient to mediate Golgi residency. ERD2 C-terminus replacement by a canonical KKXX sequence, caused biological inactivity and exclusive ER localisation. Together, the data suggest that Golgi-residency and biological activity are directly linked, which argues strongly against the typical receptor-recycling model that has been accepted for so long. Interestingly, I found an astonishingly high degree of conservation of the receptor amongst eukaryotes, and only the receptor from few species were unable to mediate ER retention of soluble ligands in plants. Combined assays provided the experimental platform to classify further ERD2 mutants into three different functional classes. Finally, I generated further data suggesting the ERD2 may have a role in vacuolar transport and protein turnover. I conclude from my work that the classical recycling model for ERD2-mediated accumulation of soluble proteins in the ER may have to be challenged in the future and that much is to be discovered about the ER-Golgi interface in eukaryotes

HIDRÓLISE ENZIMÁTICA DA LACTOSE DE PERMEADO DE SORO

The whey permeate is the residual of the concentration process of the whey proteins by ultrafiltration method. It contains important nutrients such as lactose, minerals and some proteins and lipids. It is without an ending industrial waste that causes serious damage to the environment. For its full use the lactose must be hydrolyzed to enable its consumption by intolerant people. The enzymatic hydrolysis by lactase (β-galactosidase) of Kluyveromyces lactis yeast is a safe method that does not compromise the integrity of other nutrients, enabling further use of the permeate as a raw material. This study aimed to perform tests of enzymatic hydrolysis of lactose in whey permeate formulations in a concentration of 0.2%, 0.7% and 1% at 30, 60 and 90 minutes with pH 6.3 medium and 37 °C. The reactions were monitored by high performance liquid chromatography which showed that the enzyme concentration of 0.7% at time 30 minutes formulations became safe for consumption by lactose intolerant people, according to minimum levels established by law.O permeado de soro é o resíduo do processo de concentração das proteínas do soro pelo método de ultrafiltração. Contém nutrientes importantes, como lactose, minerais e traços de proteínas e lipídeos. É um resíduo sem fim industrial estabelecido que causa sérios danos ao meio ambiente. Para que o seu aproveitamento seja integral e para permitir seu consumo por pessoas intolerantes ao açúcar do leite é preciso que a lactose seja hidrolisada. A hidrólise enzimática pela lactase (β-galactosidase) do fungo Kluyveromyces lactis é um método seguro e que não compromete a integridade dos demais nutrientes, permitindo assim o posterior uso do permeado como matériaprima. Este trabalho objetivou realizar ensaios de hidrólise enzimática da lactose de formulações de permeado de soro, em concentração de 0,2%, 0,7% e 1% nos tempos 30, 60 e 90 minutos com o pH do meio de 6,3 e temperatura de 37 ºC. As reações foram acompanhadas por cromatografia líquida de alta eficiência que mostrou que a partir da concentração enzimática de 0,7% no tempo de 30 minutos, as formulações se tornaram seguras para o consumo de intolerantes à lactose, de acordo com níveis mínimos estabelecidos pela legislação