RESUMO
Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5'-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G-2A-1G+1U+2 motif of the 5'-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5'-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5'-splice sites, but also identify common pharmacophores required for modulating 5'-splice site selection with small molecules.
Assuntos
Desenho de Fármacos , Sítios de Splice de RNA , Splicing de RNA , Humanos , Compostos Azo , Modelos Moleculares , Conformação de Ácido Nucleico , Pirimidinas , Splicing de RNA/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a ß-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.
Assuntos
Modelos Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteína Tumoral 1 Controlada por Tradução , Apoptose/genética , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ligação Proteica/genética , Humanos , Sítios de Ligação , Estrutura Quaternária de ProteínaRESUMO
O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 µg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.
Assuntos
Placenta , Proteínas , Humanos , Feminino , Gravidez , Placenta/metabolismo , Proteínas/metabolismo , Fosforilação , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change.
Assuntos
Ecossistema , Água Subterrânea , Biodiversidade , Água Doce , Poluição AmbientalRESUMO
U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon-intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.
Assuntos
Ribonucleoproteína Nuclear Pequena U1/química , Sítios de Ligação , Ligantes , Espectroscopia de Ressonância Magnética , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismoRESUMO
In Eukarya, immature mRNA transcripts (pre-mRNA) often contain coding sequences, or exons, interleaved by non-coding sequences, or introns. Introns are removed upon splicing, and further regulation of the retained exons leads to alternatively spliced mRNA. The splicing reaction requires the stepwise assembly of the spliceosome, a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs). This review focuses on the early stage of spliceosome assembly, when U1 snRNP defines each intron 5'-splice site (5'ss) in the pre-mRNA. We first introduce the splicing reaction and the impact of alternative splicing on gene expression regulation. Thereafter, we extensively discuss splicing descriptors that influence the 5'ss selection by U1 snRNP, such as sequence determinants, and interactions mediated by U1-specific proteins or U1 small nuclear RNA (U1 snRNA). We also include examples of diseases that affect the 5'ss selection by U1 snRNP, and discuss recent therapeutic advances that manipulate U1 snRNP 5'ss selectivity with antisense oligonucleotides and small-molecule splicing switches.
Assuntos
Precursores de RNA , Ribonucleoproteína Nuclear Pequena U1 , Processamento Alternativo , Precursores de RNA/genética , Sítios de Splice de RNA , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismoRESUMO
The evolutionary origin of the striking genome size variations found in eukaryotes remains enigmatic. The effective size of populations, by controlling selection efficacy, is expected to be a key parameter underlying genome size evolution. However, this hypothesis has proved difficult to investigate using empirical data sets. Here, we tested this hypothesis using 22 de novo transcriptomes and low-coverage genomes of asellid isopods, which represent 11 independent habitat shifts from surface water to resource-poor groundwater. We show that these habitat shifts are associated with higher transcriptome-wide [Formula: see text] After ruling out the role of positive selection and pseudogenization, we show that these transcriptome-wide [Formula: see text] increases are the consequence of a reduction in selection efficacy imposed by the smaller effective population size of subterranean species. This reduction is paralleled by an important increase in genome size (25% increase on average), an increase also confirmed in subterranean decapods and mollusks. We also control for an adaptive impact of genome size on life history traits but find no correlation between body size, or growth rate, and genome size. We show instead that the independent increases in genome size measured in subterranean isopods are the direct consequence of increasing invasion rates by repeat elements, which are less efficiently purged out by purifying selection. Contrary to selection efficacy, polymorphism is not correlated to genome size. We propose that recent demographic fluctuations and the difficulty of observing polymorphism variation in polymorphism-poor species can obfuscate the link between effective population size and genome size when polymorphism data are used alone.
Assuntos
Especiação Genética , Tamanho do Genoma , Isópodes/genética , Filogenia , Seleção Genética , Animais , Decápodes/classificação , Decápodes/genética , Sequenciamento de Nucleotídeos em Larga Escala , Isópodes/classificação , Repetições de Microssatélites , Moluscos/classificação , Moluscos/genética , Polimorfismo Genético , TranscriptomaRESUMO
The rate of molecular evolution varies widely among species. Life history traits (LHTs) have been proposed as a major driver of these variations. However, the relative contribution of each trait is poorly understood. Here, we test the influence of metabolic rate (MR), longevity, and generation time (GT) on the nuclear and mitochondrial synonymous substitution rates using a group of isopod species that have made multiple independent transitions to subterranean environments. Subterranean species have repeatedly evolved a lower MR, a longer lifespan and a longer GT. We assembled the nuclear transcriptomes and the mitochondrial genomes of 13 pairs of closely related isopods, each pair composed of one surface and one subterranean species. We found that subterranean species have a lower rate of nuclear synonymous substitution than surface species whereas the mitochondrial rate remained unchanged. We propose that this decoupling between nuclear and mitochondrial rates comes from different DNA replication processes in these two compartments. In isopods, the nuclear rate is probably tightly controlled by GT alone. In contrast, mitochondrial genomes appear to replicate and mutate at a rate independent of LHTs. These results are incongruent with previous studies, which were mostly devoted to vertebrates. We suggest that this incongruence can be explained by developmental differences between animal clades, with a quiescent period during female gametogenesis in mammals and birds which imposes a nuclear and mitochondrial rate coupling, as opposed to the continuous gametogenesis observed in most arthropods.
Assuntos
Evolução Molecular , Genoma Mitocondrial , Isópodes/genética , Características de História de Vida , Animais , Replicação do DNA , Ecossistema , Transporte de Elétrons , Isópodes/metabolismo , Isópodes/efeitos da radiação , Biossíntese de Proteínas , Seleção GenéticaRESUMO
The species concept is the cornerstone of biodiversity science, and any paradigm shift in the delimitation of species affects many research fields. Many biologists now are embracing a new "species" paradigm as separately evolving populations using different delimitation criteria. Individual criteria can emerge during different periods of speciation; some may never evolve. As such, a paradigm shift in the species concept relates to this inherent heterogeneity in the speciation process and species category-which is fundamentally overlooked in biodiversity research. Cryptic species fall within this paradigm shift: they are continuously being reported from diverse animal phyla but are poorly considered in current tests of ecological and evolutionary theory. The aim of this review is to integrate cryptic species in biodiversity science. In the first section, we address that the absence of morphological diversification is an evolutionary phenomenon, a "process" counterpart to the long-studied mechanisms of morphological diversification. In the next section regarding taxonomy, we show that molecular delimitation of cryptic species is heavily biased towards distance-based methods. We also stress the importance of formally naming of cryptic species for better integration into research fields that use species as units of analysis. Finally, we show that incorporating cryptic species leads to novel insights regarding biodiversity patterns and processes, including large-scale biodiversity assessments, geographic variation in species distribution and species coexistence. It is time for incorporating multicriteria species approaches aiming to understand speciation across space and taxa, thus allowing integration into biodiversity conservation while accommodating for species uncertainty.
Assuntos
Especiação Genética , Anfípodes/fisiologia , Animais , Biodiversidade , Bases de Dados como Assunto , Filogenia , Especificidade da EspécieRESUMO
The field of stoichiogenomics aims at understanding the influence of nutrient limitations on the elemental composition of the genome, transcriptome, and proteome. The 20 amino acids and the 4 nt differ in the number of nutrients they contain, such as nitrogen (N). Thus, N limitation shall theoretically select for changes in the composition of proteins or RNAs through preferential use of N-poor amino acids or nucleotides, which will decrease the N-budget of an organism. While these N-saving mechanisms have been evidenced in microorganisms, they remain controversial in multicellular eukaryotes. In this study, we used 13 surface and subterranean isopod species pairs that face strongly contrasted N limitations, either in terms of quantity or quality. We combined in situ nutrient quantification and transcriptome sequencing to test if N limitation selected for N-savings through changes in the expression and composition of the transcriptome and proteome. No evidence of N-savings was found in the total N-budget of transcriptomes or proteomes or in the average protein N-cost. Nevertheless, subterranean species evolving in N-depleted habitats displayed lower N-usage at their third codon positions. To test if this convergent compositional change was driven by natural selection, we developed a method to detect the strand-asymmetric signature that stoichiogenomic selection should leave in the substitution pattern. No such signature was evidenced, indicating that the observed stoichiogenomic-like patterns were attributable to nonadaptive processes. The absence of stoichiogenomic signal despite strong N limitation within a powerful phylogenetic framework casts doubt on the existence of stoichiogenomic mechanisms in metazoans.
Assuntos
Isópodes/genética , Isópodes/metabolismo , Nitrogênio/deficiência , Nitrogênio/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Ecossistema , Nucleotídeos/genética , Nucleotídeos/metabolismo , Filogenia , Proteoma , Seleção Genética , TranscriptomaRESUMO
A key challenge for biologists is to document and explain global patterns of diversification in a wide range of environments. Here, we explore patterns of continental-scale diversification in a groundwater species-rich clade, the superfamily Aselloidea (Pancrustacea: Isopoda). Our analyses supported a constant diversification rate during most of the course of Aselloidea evolution, until 4-15 Ma when diversification rates started to decrease. This constant accumulation of lineages challenges the view that groundwater species diversification in temperate regions might have been primarily driven by major changes in physical environment leading to the extinction of surface populations and subsequent synchronous isolation of multiple groundwater populations. Rather than acting synchronously over broad geographic regions, factors causing extinction of surface populations and subsequent reproductive isolation of groundwater populations may act in a local and asynchronous manner, thereby resulting in a constant speciation rate over time. Our phylogeny also revealed several cases of parapatric distributions among closely related surface-water and groundwater species suggesting that species diversification could also arise from a process of disruptive selection along the surface-subterranean environmental gradient. Our results call for re-evaluating the spatial scale and timing of factors causing diversification events in groundwater.
Assuntos
Evolução Molecular , Isópodes/classificação , Isópodes/genética , Animais , Teorema de Bayes , Evolução Biológica , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Europa (Continente) , Evolução Planetária , Especiação Genética , Água Subterrânea , Isópodes/metabolismo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Estados UnidosRESUMO
Isopods are a diverse group of crustaceans, that inhabit various environments, including terrestrial, freshwater, and marine, both on the surface and in the underground. The biological mechanisms underlying their wide range of adaptations to diverse ecological niches remain elusive. In order to unravel the molecular basis of their adaptability, we generated a comprehensive RNAseq dataset comprising 11 isopod species belonging to the three different suborders: freshwater Asellota, marine, brackish and freshwater Sphaeromatidea, and terrestrial Oniscidea, with representatives from families Asellidae, Sphaeromatidae, and Trichoniscidae, respectively. Representatives of each family were collected from both cave and surface environments, representing at least three independent cave colonization events. Three biological replicates were sequenced from each species to ensure data robustness. The 11 high-quality RNAseq datasets will serve as a valuable resource for understanding cave-specific adaptations, comparative and functional genomics, ecological annotation as well as aid in conservation efforts of these non-model organisms. Importantly, transcriptomes of eight featured species have been made publicly accessible for the first time.
Assuntos
Cavernas , Isópodes , Transcriptoma , Isópodes/genética , AnimaisRESUMO
In eukaryotic cells, RNA splicing is crucial for gene expression. Dysregulation of this process can result in incorrect mRNA processing, leading to aberrant gene expression patterns. Such abnormalities are implicated in many inherited diseases and cancers. Historically, antisense oligonucleotides, which bind to specific RNA targets, have been used to correct these splicing abnormalities. Despite their high specificity of action, these oligonucleotides have drawbacks, such as lack of oral bioavailability and the need for chemical modifications to enhance cellular uptake and stability. As a result, recent efforts focused on the development of small organic molecules that can correct abnormal RNA splicing event under disease conditions. This review discusses known and potential targets of these molecules, including RNA structures, trans-acting splicing factors, and the spliceosome - the macromolecular complex responsible for RNA splicing. We also rely on recent advances to discuss therapeutic applications of RNA-targeting small molecules in splicing correction. Overall, this review presents an update on strategies for RNA splicing modulation, emphasizing the therapeutic promise of small molecules.
RESUMO
Ribonucleases (RNases) are ubiquitous enzymes that process or degrade RNA, essential for cellular functions and immune responses. The EndoU-like superfamily includes endoribonucleases conserved across bacteria, eukaryotes, and certain viruses, with an ancient evolutionary link to the ribonuclease A-like superfamily. Both bacterial EndoU and animal RNase A share a similar fold and function independently of cofactors. In contrast, the eukaryotic EndoU catalytic domain requires divalent metal ions for catalysis, possibly due to an N-terminal extension near the catalytic core. In this study, we used biophysical and computational techniques along with in vitro assays to investigate the calcium-dependent activation of human EndoU. We determined the crystal structure of EndoU bound to calcium and found that calcium binding remote from the catalytic triad triggers water-mediated intramolecular signaling and structural changes, activating the enzyme through allostery. Calcium-binding involves residues from both the catalytic core and the N-terminal extension, indicating that the N-terminal extension interacts with the catalytic core to modulate activity in response to calcium. Our findings suggest that similar mechanisms may be present across all eukaryotic EndoUs, highlighting a unique evolutionary adaptation that connects endoribonuclease activity to cellular signaling in eukaryotes.
RESUMO
Transition to novel environments, such as groundwater colonization by surface organisms, provides an excellent research ground to study phenotypic evolution. However, interspecific comparative studies on evolution to groundwater life are few because of the challenge in assembling large ecological and molecular resources for species-rich taxa comprised of surface and subterranean species. Here, we make available to the scientific community an operational set of working tools and resources for the Asellidae, a family of freshwater isopods containing hundreds of surface and subterranean species. First, we release the World Asellidae database (WAD) and its web application, a sustainable and FAIR solution to producing and sharing data and biological material. WAD provides access to thousands of species occurrences, specimens, DNA extracts and DNA sequences with rich metadata ensuring full scientific traceability. Second, we perform a large-scale dated phylogenetic reconstruction of Asellidae to support phylogenetic comparative analyses. Of 424 terminal branches, we identify 34 pairs of surface and subterranean species representing independent replicates of the transition from surface water to groundwater. Third, we exemplify the usefulness of WAD for documenting phenotypic shifts associated with colonization of subterranean habitats. We provide the first phylogenetically controlled evidence that body size of males decreases relative to that of females upon groundwater colonization, suggesting competition for rare receptive females selects for smaller, more agile males in groundwater. By making these tools and resources widely accessible, we open up new opportunities for exploring how phenotypic traits evolve in response to changes in selective pressures and trade-offs during groundwater colonization.
Assuntos
Isópodes , Animais , Filogenia , Isópodes/genética , Ecossistema , DNA , Sequência de BasesRESUMO
Non-nutritive sweeteners (NNS) are popular sugar replacements used in foods, beverages, and medications. Although NNS are considered safe by regulatory organizations, their effects on physiological processes such as detoxification are incompletely understood. Previous studies revealed that the NNS sucralose (Sucr) altered P-glycoprotein (PGP) expression in rat colon. We also demonstrated that early-life exposure to NNS Sucr and acesulfame potassium (AceK) compromises mouse liver detoxification. Building upon these initial discoveries, we investigated the impact of AceK and Sucr on the PGP transporter in human cells to assess whether NNS influence its key role in cellular detoxification and drug metabolism. We showed that AceK and Sucr acted as PGP inhibitors, competing for the natural substrate-binding pocket of PGP. Most importantly, this was observed after exposure to concentrations of NNS within expected levels from common foods and beverage consumption. This may suggest risks for NNS consumers, either when taking medications that require PGP as the primary detoxification transporter or during exposure to toxic compounds.
Assuntos
Adoçantes não Calóricos , Tiazinas , Ratos , Humanos , Animais , Camundongos , Adoçantes não Calóricos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATPRESUMO
Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy but requires high levels of the adaptor protein DCAF15. Consequently, novel approaches to deplete RBM39 in an DCAF15-independent manner are required. Here, we uncover that RBM39 autoregulates via the inclusion of a poison exon into its own pre-mRNA and identify the cis-acting elements that govern this regulation. We also determine the NMR solution structures of RBM39's tandem RNA recognition motifs (RRM1 and RRM2) bound to their respective RNA targets, revealing how RRM1 recognises RNA stem loops whereas RRM2 binds specifically to single-stranded N(G/U)NUUUG. Our results support a model where RRM2 selects the 3'-splice site of a poison exon and the RRM3 and RS domain stabilise the U2 snRNP at the branchpoint. Our work provides molecular insights into RBM39-dependent 3'-splice site selection and constitutes a solid basis to design alternative anti-cancer therapies.
Assuntos
Neoplasias , Splicing de RNA , Splicing de RNA/genética , Motivos de Ligação ao RNA , Sítios de Splice de RNA , Homeostase , Fatores de Processamento de RNA/genética , Neoplasias/genéticaRESUMO
TCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported inâ vitro by SPR experiments to be in the â¼30-50â µM Kd range (Amson etâ al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent â¼mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature.
Assuntos
Antineoplásicos/farmacologia , Sertralina/farmacologia , Tioridazina/farmacologia , Proteína Tumoral 1 Controlada por Tradução/antagonistas & inibidores , Antineoplásicos/química , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Molecular , Sertralina/química , Relação Estrutura-Atividade , Tioridazina/química , Proteína Tumoral 1 Controlada por Tradução/isolamento & purificação , Proteína Tumoral 1 Controlada por Tradução/metabolismoRESUMO
Post-translational modifications (PTMs) are ubiquitous and essential for protein function and signaling, motivating the need for sustainable benefit and open models of web databases. Highly conserved O-GlcNAcylation is a case example of one of the most recently discovered PTMs, investigated by a growing community. Historically, details about O-GlcNAcylated proteins and sites were dispersed across literature and in non-O-GlcNAc-focused, rapidly outdated or now defunct web databases. In a first effort to fill the gap, we recently published a human O-GlcNAcome catalog with a basic web interface. Based on the enthusiasm generated by this first resource, we extended our O-GlcNAcome catalog to include data from 42 distinct organisms and released the O-GlcNAc Database v1.2. In this version, more than 14 500 O-GlcNAcylated proteins and 11 000 O-GlcNAcylation sites are referenced from the curation of 2200 publications. In this article, we also present the extensive features of the O-GlcNAc Database, including the user-friendly interface, back-end and client-server interactions. We particularly emphasized our workflow, involving a mostly automatized and self-maintained database, including machine learning approaches for text mining. We hope that this software model will be useful beyond the O-GlcNAc community, to set up new smart, scientific online databases, in a short period of time. Indeed, this database system can be administrated with little to no programming skills and is meant to be an example of a useful, sustainable and cost-efficient resource, which exclusively relies on free open-source software elements (www.oglcnac.mcw.edu).