Spectroscopic studies of the conformational stability and ligand binding properties of calmodulin

In this work the conformational stability of calmodulin (CaM) and its tryptic fragments has been investigated using optical spectroscopic techniques such as far-UV circular dichroism, fluorescence, and absorption spectroscopy. CaM is a ubiquitous eukaryotic calcium binding protein that consists of two structurally similar globular domains connected by a flexible linker, and each containing a pair of helix-loop-helix calcium binding motifs. The results of chemical and thermal denaturation experiments show that the stability of CaM and its isolated domains in the absence of calcium is relatively low and is strongly dependent on ionic strength and temperature. This raises the question of how apo-CaM is stabilised in vivo at resting calcium concentrations. In the presence of ligands such as calcium, magnesium, and target peptides, the stability of CaM is greatly enhanced, as predicted by the ligand binding theory. The extent of the stabilising effect depends on the free ligand concentration and on the affinity of the native and denatured states of CaM for the ligand. The interactions of CaM with magnesium have been investigated and the results show that magnesium competes with calcium for the EF- hand sites and reduces the affinity of Ca2+ -CaM for targets. As a consequence, magnesium amplifies the intrinsic differences in affinity of the N- and C-domains for calcium and for target sequences. Thus, CaM is extremely sensitive to general environmental conditions as well as to specific ligand interactions. Chemical denaturation studies also show that the behaviour of GuHCl is very complex, owing to its ionic strength contribution and to competition with Ca2+ -binding. Therefore urea may be more appropriate in the study of the stability of calmodulin and of proteins that bind metal ions or that are sensitive to changes in the ionic strength of the solution. Finally, there is experimental evidence that the stability of the isolated domains of CaM is significantly different from that of the domains in the intact protein. This suggests the presence of inter-domain interactions during the process of unfolding. This observation is confirmed by the analysis of the stability of the [beta]-sheet mutants of CaM, which shows that the structural and Ca2+-binding properties of the non-mutated domain can be affected by the mutation on the other domain

Using EEG to Understand the Effects of Top-Down Processing on Speech Perception

Cognitive psychologists believe that our brain not only interprets bottom-up information received from sensory input, but prior knowledge can also change what we hear in a top-down manner (Getz & Toscano, 2019). In this study, we were interested in collecting electroencephalography (EEG) data to determine how strongly top-down processing impacts the perception process. Our stimuli consisted of common word pairs (e.g., bunk beds, amusement park) in which target words were manipulated to have varying voice onset times (VOTs). The sounds /b/ and /p/ exist on a VOT continuum, with /b/ having a short VOT (voiced) and /p/ having a longer VOT (voiceless); /d/ and /t/ follow the same pattern. During the experiment, participants determined the starting sound (b, d, p, t) of the second word in each pair. The first word was either an association prime or neutral prime. We began with a behavioral pilot test, investigating how various top-down factors would affect reaction times. We varied the word frequency, neighborhood density, and lexical status of the primes. We found that responses were most impacted by lexical status, meaning participants were more likely to perceive ambiguous targets as words (rather than non-words). For associated primes, responses differed based on expected voicing (an ambiguous VOT between b/p was perceived as /b/ in the context of bunk BEDS, but as /p/ in amusement PARK). We are currently using EEG to track brain voltage fluctuations and are conducting ERP analysis to understand the time course of top-down information\u27s influence on speech processing

Lipid profile and growth of black soldier flies (Hermetia illucens, Stratiomyidae) reared on by‐products from different food chains

BACKGROUND The total amount of bio‐waste produced annually in the EU by the food and beverage chains is estimated at 37 Mtons. The possibility to use insects for the valorization of by‐products from these value chains may represent a sustainable solution. This study aims at investigating the by‐products obtained from different food chains for the rearing of black soldier fly prepupae to evaluate lipid content and profile and outline its possible applications. The substrates used in this experiment were: (i) industrial by‐products (brewery spent grains, cow's milk whey, grape stalks, and tomato peels and seeds) and (ii) by‐products from retailers (bread dough, fish scraps, and spent coffee ground). Fat extracted from prepupae using an adjusted Folch method was utilized for total lipid content and fatty acids profile. RESULTS Best larval performances were obtained from beer (0.22 gweight per prepupa), tomato (0.19 gweight per prepupa), and cheese (0.14 gweight per prepupa) food‐chain by‐products. The extremely different composition of the substrate was reflected in the differentiated lipid profile of black soldier fly prepupae and in a range of ratios between unsaturated and saturated fatty acids comprised from 0.37 for cow's milk way to 1.34 for tomato peels and seeds. CONCLUSION The high content and type of lipids, together with the proteins, and chitin extracted from prepupae are high‐value bio‐based products that could be used in the feed/food industry or for the development of innovative biomaterials, such as biodiesel. These results suggest that food chain by‐products are the best candidate for insect‐bioconversion purposes

Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases

Abstract RING-between-RING (RBR) ubiquitin ligases work with multiple E2 enzymes and function through an E3-ubiquitin thioester intermediate. The RBR module comprises three domains, RING1, IBR and RING2 that collaborate to transfer ubiquitin from the E2~Ub conjugate, recognised by RING1, onto a catalytic cysteine in RING2 and finally onto the substrate in a multi-step reaction. Recent studies have shown that RING1 domains bind E2~Ub conjugates in an open conformation to supress ubiquitin transfer onto lysine residues and promote formation of the E3 thioester intermediate. However, how the nature of the E2 influences the ubiquitin transfer process is currently unclear. We report here a detailed characterization of the RBR/E2-conjugate recognition step that indicates that this mechanism depends on the nature of the E2 enzyme and differs between UbcH5 and UbcH7. In the case of UbcH5~Ub an interaction with ubiquitin is necessary to stabilize the transfer complex while recognition of UbcH7~Ub is driven primarily by E2-RING1 contacts. Furthermore our analysis suggests that RBRs, in isolation and in complex with ubiquitin-loaded E2s, are dynamic species and that their intrinsic flexibility might be a key aspect of their catalytic mechanism

A malaria parasite subtilisin propeptide-like protein is a potent inhibitor of the egress protease SUB1.

Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences

The Interplay between PolyQ and Protein Context Delays Aggregation by Forming a Reservoir of Protofibrils

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway

Solution structure of polyglutamine tracts in GST-polyglutamine fusion proteins

AbstractAggregation of expanded polyglutamine (polyQ) seems to be the cause of various genetic neurodegenerative diseases. Relatively little is known as yet about the polyQ structure and the mechanism that induces aggregation. We have characterised the solution structure of polyQ in a proteic context using a model system based on glutathione S-transferase fusion proteins. A wide range of biophysical techniques was applied. For the first time, nuclear magnetic resonance was used to observe directly and selectively the conformation of polyQ in the pathological range. We demonstrate that, in solution, polyQs are in a random coil conformation. However, under destabilising conditions, their aggregation behaviour is determined by the polyQ length

Gestión y planificación de espacios periurbanos. Abordaje prospectivo en Corral de Bustos Ifflinger (Córdoba)

La temática periurbana ha cobrado relevancia debido a las tensiones y/o a las oportunidades que pudieran generarse en torno a ella. La producción agropecuaria en los espacios periurbanos -sobre todo en zonas de producción agrícola convencional de commodities- sumado a la expansión urbana sin una adecuada planificación territorial, suelen crear disputas y conflictos que afectan el orden social, económico, productivo, político, ambiental y urbanístico del lugar. Los periurbanos también cumplen múltiples funciones: generan bienes y servicios, renta y empleo; aportan a la seguridad y soberanía alimentaria local, regional y/o nacional; ofician de límite verde, barrera y/o buffer; tienen una función paisajística; entre otros servicios ecosistémicos. Por ello, requieren ser gestionados y planificados con la participación de todos los actores sociales del territorio. En el sudeste de la provincia de Córdoba, el Instituto Nacional de Tecnología Agropecuaria (INTA), la Universidad Nacional de Villa María (UNVM), y el Municipio de Corral de Bustos Ifflinger, trabajan articuladamente para profundizar el análisis del periurbano de la localidad, con el fin de orientar un proceso participativo e integral, que permita a los actores del territorio tomar decisiones en el presente para contribuir a un futuro deseado. Se emplea la Prospectiva Territorial como herramienta metodológica de abordaje, con un horizonte temporal al año 2030, para generar marcos de referencia compartidos, sustentando la planificación y gestión territorial a largo plazo, generando compromisos y abonando al diálogo permanente entre los actores sociales del territorio. En este artículo nos proponemos presentar la propuesta de trabajo interinstitucional para el abordaje prospectivo del periurbano de Corral de Bustos Ifflinger, el marco metodológico y los avances parciales relacionados al diagnóstico prospectivo (primera fase del proceso).EEA Marcos JuárezFil: Bodrero, Mercedes. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina.Fil: Girardo, Silvana. Universidad del Gran Rosario. Rosario; ArgentinaFil: Madoery, Ormando Aníbal. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina.Fil: Guzmán, Ana. Universidad Nacional de Villa María; Argentina.Fil: Cañete, Valentín. Municipalidad de Corral de Bustos-Ifflinger; Argentina.Fil: Escolá, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina.Fil: Masino, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Agencia de Extensión Rural Corral de Bustos; Argentina.Fil: Tolchinsky, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina.Fil: Gadban, Laura Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina.Fil: Vitale Gutiérrez, Javier Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro Regional Mendoza-San Juan, Argentina.Fil: Defagot, Melisa Ana Velia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina.Fil: Muñoz, Sebastián. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies