Automated Collective Variable Discovery for MFSD2A transporter from molecular dynamics simulations.

Biomolecules often exhibit complex free energy landscapes in which long-lived metastable states are separated by large energy barriers. Overcoming these barriers to robustly sample transitions between the metastable states with classical molecular dynamics (MD) simulations presents a challenge. To circumvent this issue, collective variable (CV)-based enhanced sampling MD approaches are often employed. Traditional CV selection relies on intuition and prior knowledge of the system. This approach introduces bias, which can lead to incomplete mechanistic insights. Thus, automated CV detection is desired to gain a deeper understanding of the system/process. Analysis of MD data with various machine learning algorithms, such as Principal Component Analysis (PCA), Support Vector Machine (SVM), and Linear Discriminant Analysis (LDA)-based approaches have been implemented for automated CV detection. However, their performance has not been systematically evaluated on structurally and mechanistically complex biological systems. Here, we applied these methods to MD simulations of the MFSD2A (Major Facilitator Superfamily Domain 2A) lysolipid transporter in multiple functionally relevant metastable states with the goal of identifying optimal CVs that would structurally discriminate these states. Specific emphasis was on the automated detection and interpretive power of LDA-based CVs. We found that LDA methods, which included a novel gradient descent-based multiclass harmonic variant, termed GDHLDA, we developed here, outperform PCA in class separation, exhibiting remarkable consistency in extracting CVs critical for distinguishing metastable states. Furthermore, the identified CVs included features previously associated with conformational transitions in MFSD2A. Specifically, conformational shifts in transmembrane helix 7 and in residue Y294 on this helix emerged as critical features discriminating the metastable states in MFSD2A. This highlights the effectiveness of LDA-based approaches in automatically extracting from MD trajectories CVs of functional relevance that can be used to drive biased MD simulations to efficiently sample conformational transitions in the molecular system.

Disentangling Unusual Catalytic Properties and the Role of the [4Fe-4S] Cluster of Three Endonuclease III from the Extremophile D. radiodurans.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities.

Dynamic Profiling of ß-Coronavirus 3CL Mpro Protease Ligand-Binding Sites.

ß-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across ß-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications.

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.

Insights into the transcriptional and post-transcriptional regulation of the rice SUMOylation machinery and into the role of two rice SUMO proteases.

BACKGROUND: SUMOylation is an essential eukaryotic post-translation modification that, in plants, regulates numerous cellular processes, ranging from seed development to stress response. Using rice as a model crop plant, we searched for potential regulatory points that may influence the activity of the rice SUMOylation machinery genes. RESULTS: We analyzed the presence of putative cis-acting regulatory elements (CREs) within the promoter regions of the rice SUMOylation machinery genes and found CREs related to different cellular processes, including hormone signaling. We confirmed that the transcript levels of genes involved in target-SUMOylation, containing ABA- and GA-related CREs, are responsive to treatments with these hormones. Transcriptional analysis in Nipponbare (spp. japonica) and LC-93-4 (spp. indica), showed that the transcript levels of all studied genes are maintained in the two subspecies, under normal growth. OsSUMO3 is an exceptional case since it is expressed at low levels or is not detectable at all in LC-93-4 roots and shoots, respectively. We revealed post-transcriptional regulation by alternative splicing (AS) for all genes studied, except for SUMO coding genes, OsSIZ2, OsOTS3, and OsELS2. Some AS forms have the potential to alter protein domains and catalytic centers. We also performed the molecular and phenotypic characterization of T-DNA insertion lines of some of the genes under study. Knockouts of OsFUG1 and OsELS1 showed increased SUMOylation levels and non-overlapping phenotypes. The fug1 line showed a dwarf phenotype, and significant defects in fertility, seed weight, and panicle architecture, while the els1 line showed early flowering and decreased plant height. We suggest that OsELS1 is an ortholog of AtEsd4, which was also supported by our phylogenetic analysis. CONCLUSIONS: Overall, we provide a comprehensive analysis of the rice SUMOylation machinery and discuss possible effects of the regulation of these genes at the transcriptional and post-transcriptional level. We also contribute to the characterization of two rice SUMO proteases, OsELS1 and OsFUG1.

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose-phosphate synthase and is required for a proper cold stress response.

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose-phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

Synthetic Red Blood Cell-Specific Glycolytic Intermediate 2,3-Diphosphoglycerate (2,3-DPG) Inhibits Plasmodium falciparum Development In Vitro.

Mechanisms of malaria parasite interaction with its host red blood cell may provide potential targets for new antimalarial approaches. Pyruvate kinase deficiency has been associated with resistance to malaria in both experimental models and population studies. Two of the major pyruvate kinase deficient-cell disorders are the decrease in ATP and the increase in 2,3-biphosphoglycerate (2,3-BPG) concentration. High levels of this metabolite, only present in mammalian red blood cell, has an inhibitory effect on glycolysis and we hypothesized that its accumulation may also be harmful to the parasite and be involved in the mechanism of protection provided by that enzymopathy. We examined the effect of a synthetic form, 2,3-DPG, on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. Results showed an impairment of parasite growth with a direct effect on parasite maturation as significant lower progeny emerged from parasites that were submitted to 2,3-DPG. Further, adding the compound to the culture medium did not result in any effect on the host cell, but instead the metabolic profile of an infected cell became closer to that of a non-infected cell.

Exploring the regulatory levels of SUMOylation to increase crop productivity.

SUMOylation is an essential post-translational modification that affects several cellular processes, from gene replication to stress response. Studies using the SUMO (de)conjugation machinery have provided evidence regarding its potential to improve crop performance and productivity under normal and adverse conditions. However, the pleiotropic effect of SUMOylation can be a disadvantage in both situations, especially when considering unpredictable environmental conditions caused by climate changes. Here, we discuss the pleiotropic effects caused by disrupting the SUMOylation machinery, and new strategies that may help to overcome pleiotropy. We propose exploring the several regulatory levels of SUMOylation recently revealed, including transcriptional, post-transcriptional regulation by alternative splicing, and post-translational modifications. These new findings may provide valuable tools to increase crop productivity.

The interplay between Mn and Fe in Deinococcus radiodurans triggers cellular protection during paraquat-induced oxidative stress.

The bacterium Deinococcus radiodurans is highly resistant to several stress conditions, such as radiation. According to several reports, manganese plays a crucial role in stress protection, and a high Mn/Fe ratio is essential in this process. However, mobilization of manganese and iron, and the role of DNA-binding-proteins-under-starved-conditions during oxidative-stress remained open questions. We used synchrotron-based X-ray fluorescence imaging at nano-resolution to follow element-relocalization upon stress, and its dependency on the presence of Dps proteins, using dps knockout mutants. We show that manganese, calcium, and phosphorus are mobilized from rich-element regions that resemble electron-dense granules towards the cytosol and the cellular membrane, in a Dps-dependent way. Moreover, iron delocalizes from the septum region to the cytoplasm affecting cell division, specifically in the septum formation. These mechanisms are orchestrated by Dps1 and Dps2, which play a crucial role in metal homeostasis, and are associated with the D. radiodurans tolerance against reactive oxygen species.

Expression, purification and crystallization of MnSOD from Arabidopsis thaliana.

Manganese superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme. MnSOD plays an important role in plant tolerance to abiotic stress and is a target candidate for increasing stress tolerance in crop plants. Although the structure and kinetic parameters of MnSODs from several organisms have been determined, this information is still lacking for plant MnSODs. Here, recombinant MnSOD from Arabidopsis thaliana (AtMnSOD) was expressed, purified and crystallized. A nearly complete data set could only be obtained when a total rotation range of 180° was imposed during data collection, despite the seemingly tetragonal metric of the AtMnSOD crystal diffraction. The data set extended to 1.95 Å resolution and the crystal belonged to space group P1. Molecular-replacement calculations using an ensemble of homologous SOD structures as a search model gave a unique and unambiguous solution corresponding to eight molecules in the asymmetric unit. Structural and kinetic analysis of AtMnSOD is currently being undertaken.

A morte da pessoa numa unidade de cuidados intensivos coronários: Vivências dos enfermeiros / The death of the person in a coronary intensive care unit: Nurses' experiences

Lidar com a morte é uma experiência que ninguém quer encarar. Contudo, o enfermeiro tem um papel fundamental de ajuda e apoio à pessoa e família nesta aprendizagem (Sulzbacher et al., 2009). A forma como os enfermeiros lidam com a morte depende de múltiplos fatores (Dias, 2010). Entre eles, as suas características pessoais, a sua formação, o contexto onde trabalha, ou mesmo a pessoa que morre. Numa unidade de cuidados intensivos coronários (UCIC), todos os esforços são desenvolvidos para manter a vida de quem é cuidado. Raramente se aceita a morte sem lhe ser dada luta acérrima. Conhecer as vivências dos enfermeiros perante a morte da pessoa cuidada numa UCIC, foi o objetivo deste estudo. Recorreu-se para tal a uma abordagem qualitativa, com à recolha de dados através de entrevista semiestruturada, realizada a 6 enfermeiros com um mínimo de 3 anos de experiência numa UCIC e 5 anos de experiência profissional. Os dados foram analisados com recurso à análise de conteúdo (Bardin, 2004). Da análise dos dados obtiveram-se três categorias principais: 1) "Emoções e Sentimentos", que podem ser "Ameaçadores" ("Tristeza", "Revolta", "Angústia", "Frustração", "Impotência" e "Insegurança") ou "Positivos" ("Compaixão" e "Dever cumprido"); 2) "Estratégias/Recursos para fazer face", que podem ser "Individuais" (como o "Reavaliar a situação", o "Envolvimento profissional" ou o "Encarar a morte como algo natural", entre outras) ou "Com os Outros" ("Partilha de experiências entre colegas" e "Suporte familiar"); 3) a última categoria reporta as "Dificuldades sentidas", que integram a dificuldade em "Lidar com a morte/fracasso" e em "Lidar com os próprios sentimentos". As descrições das experiências sentidas destes enfermeiros em conjunto com a reflexão e discussão realizadas, poderão proporcionar melhorias ao nível do cuidar, especialmente em contextos semelhantes. Podem resultar ainda programas de melhoria contínua, nomeadamente ao nível da preparação para a gestão do luto ou para o próprio apoio do enfermeiro enquanto pessoa cuidadora.