Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to...Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology. We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six proteins and demonstrate (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.
Antibodies are valuable biological reagents used in a wide range of discovery research, biotechnology, diagnostic and therapeutic applications. Currently, both commercial and laboratory-scale antibody production is relia...Antibodies are valuable biological reagents used in a wide range of discovery research, biotechnology, diagnostic and therapeutic applications. Currently, both commercial and laboratory-scale antibody production is reliant on expression from mammalian cells, which can be time-consuming and requires the use of specialist facilities and costly growth reagents. Here, we describe a simple, rapid and cheap method for producing and isolating functional monoclonal antibodies and antibody fragments from bacterial cells that can be used in a range of laboratory applications. This simple method only requires access to basic microbial cell culture and molecular biology equipment, making scalable in-house antibody production accessible to the global diagnostics, therapeutics and molecular bioscience research communities.
Scriptaid is a chemical compound with anti-tumoural effects due to its role as a histone deacetylase inhibitor. Despite sharing part of the chemical structure with other ligands of G-quadruplexes (G4s), the interaction o...Scriptaid is a chemical compound with anti-tumoural effects due to its role as a histone deacetylase inhibitor. Despite sharing part of the chemical structure with other ligands of G-quadruplexes (G4s), the interaction of Scriptaid with G4s has not been explored before. We synthesized Scriptaid and screened its cytotoxic activity in cellular models of colorectal cancer (CRC). We extensively evaluated its biological activity by cell cycle, immunofluorescence, qRT-PCR and Western blot experiments. To identify the G4 targets of Scriptaid, we conducted a panel of binding assays. Here, we show that Scriptaid induced cytotoxicity, cell cycle arrest and nucleolar stress in CRC cells. Moreover, Scriptaid impaired RNA polymerase I (Pol I) transcription, stabilized G4s and caused DNA damage. Finally, we disclose that these effects were attributable to the binding of Scriptaid to G4s in ribosomal DNA. In conclusion, our work reveals that a primary impact of Scriptaid on human cells is the interaction with G4s.
High cholesterol levels are associated with an increased risk of cardiovascular disease, specifically atherosclerosis, a leading cause of death worldwide. Atherosclerosis occurs when cholesterol and fat build up in plaqu...High cholesterol levels are associated with an increased risk of cardiovascular disease, specifically atherosclerosis, a leading cause of death worldwide. Atherosclerosis occurs when cholesterol and fat build up in plaques along blood vessel walls, restricting blood flow and preventing nutrients and oxygen from diffusing in and out of the bloodstream. High-density lipoprotein cholesterol (HDL) particles prevent the build-up of such plaques, removing excess cholesterol from the peripheral tissues and delivering it to the liver, where it can be removed from the body. This pathway is known as reverse cholesterol transport (RCT). Because HDL plays a key role in preventing plaque buildup, understanding how this molecule and RCT function in the body could help us develop much-needed new atherosclerosis therapies and prevention strategies. However, HDL metabolism is complex, and research on HDL has been less favoured than research investigating a much better-understood molecule, low-density lipoprotein cholesterol, as a treatment target. More specifically, the receptors involved in the process of taking up HDL within the liver and their relationships to one another, along with the mechanism of whole, or holoparticle uptake of HDL remain to be clarified. In this review, we discuss several outstanding mysteries in HDL metabolism, consider why previous clinical trials to improve cardiovascular health by modulating HDL levels have been unsuccessful and argue that understanding HDL metabolism is essential for crafting interventions to reduce cardiovascular disease risk.
The human HELQ helicase is a superfamily 2, 3'-5 helicase homologous to POLQ and RNA helicases of the Ski2-like subfamily. It is involved in diverse aspects of DNA repair and is an emerging prognosis biomarker and novel...The human HELQ helicase is a superfamily 2, 3'-5 helicase homologous to POLQ and RNA helicases of the Ski2-like subfamily. It is involved in diverse aspects of DNA repair and is an emerging prognosis biomarker and novel drug target for cancer therapy. HELQ interacts with RPA through its inherently disordered N-HELQ domain and hence is recruited to RPA-bound DNA substrates. Our study reveals a novel role for HELQ in R-loop resolution. We show in cells and that HELQ is recruited by RPA at R-loops, which are then resolved if HELQ is catalytically active as an ATPase/helicase. Furthermore, we identify a functional interaction of HELQ with XRN2, a nuclear 5' to 3' exoribonuclease, which we suggest coordinates R-loop unwinding by HELQ with RNA digestion by XRN2. Collectively, we assign a new biological function for HELQ in genome stability in metazoans through its involvement with XRN2 in R-loop metabolism.
Biotin identification (BioID) is an interactomics approach that utilizes proximity labelling to map the local interactome or proxeome of proteins within a cell. This study applies BioID to investigate proteins proximal t...Biotin identification (BioID) is an interactomics approach that utilizes proximity labelling to map the local interactome or proxeome of proteins within a cell. This study applies BioID to investigate proteins proximal to NAA60 (N-alpha-acetyltransferase 60), an N-terminal acetyltransferase (NAT) of pathological significance in human disease, characterized by its unique Golgi localization. NAA60 is known to N-terminally acetylate transmembrane proteins that present their N-terminus on the cytosolic face of the membrane, and its involvement in maintaining Golgi structure has previously been established. Using a stable cell-line expressing an NAA60-BirA* fusion protein, we isolated biotinylated proteins through streptavidin affinity purification. Mass spectrometry analysis revealed over 100 proximal partners of NAA60, enriched in proteins localized on the -side of the Golgi apparatus. High-confidence proximity interactors included golgins and GRASP proteins, essential for Golgi integrity. Considering the transmembrane nature of NAA60, the identification of biotinylated peptides inferred the topology of transmembrane protein interactors within the secretory pathway. Subsequent suborganellar localization analysis revealed a more prominent /-Golgi localization of NAA60. Our findings underscore the role of NAA60 and its interactors in maintaining Golgi structural integrity and highlight the effectiveness of BioID in generating critical protein topology data, invaluable for enhancing the prediction of protein topology within cellular compartments.
Social immunity-mediated sanitation behaviours occur in insects when microbially killed corpses are removed and/or dismembered by healthy nestmates. However, little is known concerning the chemical signals or receptor pr...Social immunity-mediated sanitation behaviours occur in insects when microbially killed corpses are removed and/or dismembered by healthy nestmates. However, little is known concerning the chemical signals or receptor proteins that mediate these responses. Here, we identify cuticular components in the eusocial red important fire ant, : behenic acid, which induces dismemberment behaviour, and oleic and ,-9,12-linoleic acids, which inhibit dismemberment in a process mediated by odorant-binding protein-15 (SiOBP15). Yeast two-hybrid screening and protein-protein interaction analyses identified the ant immunity-related proteins apolipophorin-III (SiApoLp-III) and fatty acid binding protein-5 (SiFABP5) as SiOBP15 interacting partners. SiOBP15 and SiFABP5 bound all three dismemberment-related compounds, whereas interactions between SiOBP15 and SiApoLp-III narrowed binding to behenic acid. RNAi-mediated gene expression knockdown of , or revealed that behenic acid chemoreception determines dismemberment behaviour via SiApoLp-III/SiOBP15, whereas SiOBP15 or SiOBP15/SiFABP5 recognition of linoleic acid inhibits dismemberment behaviour. These data identify a host circuit linking olfactory proteins and proteins involved in innate immunity to control the degree of sanitation behaviour elicited in response to microbial infection. We identify specific chemical cues transduced by these proteins, providing a mechanism connecting olfaction-related processes to innate immunity, host-pathogen interactions and social immunity.
This open question research article highlights mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), which have emerged as crucial cellular structures that challenge our traditional understanding of organe...This open question research article highlights mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), which have emerged as crucial cellular structures that challenge our traditional understanding of organelle function. This review highlights the critical importance of MAMs as a frontier in cell biology with far-reaching implications for health, disease and ageing. MAMs serve as dynamic communication hubs between the ER and mitochondria, orchestrating essential processes such as calcium signalling, lipid metabolism and cellular stress responses. Recent research has implicated MAM dysfunction in a wide array of conditions, including neurodegenerative diseases, metabolic disorders, cardiovascular diseases and cancer. The significant lack of biological knowledge behind MAM function emphasizes the need to study these enigmatic subcellular sites in greater detail. Key open questions include the mechanisms controlling MAM formation and disassembly, the full complement of MAM-associated proteins and how MAMs contribute to cellular decision-making and ageing processes. Advancing our understanding of MAMs through interdisciplinary approaches and cutting-edge technologies promises to reveal new insights into fundamental cellular signalling pathways and potentially lead to innovative therapeutic strategies for a range of diseases. As such, MAM research represents a critical open question in biology with the potential to transform our understanding of cellular life and human health.
Migration is a widely observed phenomenon supported by morphological, physiological and behavioural traits that vary with season and sex in many species. Recently, the genetic components underpinning migration in the mar...Migration is a widely observed phenomenon supported by morphological, physiological and behavioural traits that vary with season and sex in many species. Recently, the genetic components underpinning migration in the marmalade hoverfly (Diptera: Syrphidae) have been unpacked through detection of differentially expressed genes between migrant and non-migrant females. Males also migrate, but changing sex ratios during autumn migration, from around 50% female in northern Europe to around 90% in southern Europe, suggests males are poor long-distance fliers. To elucidate the mechanisms underpinning this sex difference, we performed morphological, physiological and transcriptomic characterization of actively migrating females and males. Both sexes show similar physiological adaptations including hyperphagia and starvation resistance, but females display higher tolerance to cold, have lower wing loading values and display a greater flight capacity. In addition, females modulate the expression of genes involved in immunity, hypoxia and longevity while suppressing hormonal pathways involved in maintaining reproductive diapause. These traits contribute to the success of female migrants and underlie the diminishing pool of males, influencing population dynamics across huge geographic areas and through the whole migratory and overwintering period.
The conserved process of centriole duplication requires the establishment of a Sas6-centred cartwheel initiated by Plk4's phosphorylation of Ana1/STIL. Subsequently, the centriole undergoes conversion to a centrosome req...The conserved process of centriole duplication requires the establishment of a Sas6-centred cartwheel initiated by Plk4's phosphorylation of Ana1/STIL. Subsequently, the centriole undergoes conversion to a centrosome requiring its radial expansion and elongation, mediated by a network requiring interactions between Cep135, Ana1/Cep295 and Asterless/Cep152. Here, we show that mutant alleles encoding overlapping N- and C-terminal parts of Ana1 are capable of intragenic complementation to rescue radial expansion. This permits the recruitment of Asl and thereby centriole duplication and mechanosensory cilia formation to restore the coordination defects of these mutants. This genetic combination also rescues centriole duplication in the male germ line but does not rescue the elongation of the triplet microtubule-containing centrioles of primary spermatocytes. Consequently, these males are coordinated but sterile. Such centriole elongation is rescued by the continuous, full-length Ana1 sequence. We define a region that when deleted within otherwise intact Ana1 does not permit primary spermatocyte centrioles to elongate but still allows recruitment of Asl. Our findings point to differing demands upon the physical organization of Ana1 for the distinct processes of radial expansion and elongation of centrioles.
Rojas-Pirela M, Andrade-Alviárez D, Rojas V
… +8 more, Marcos M, Salete-Granado D, Chacón-Arnaude M, Pérez-Nieto MÁ, Kemmerling U, Concepción JL, Michels PAM, Quiñones W
Glycolysis, present in most organisms, is evolutionarily one of the oldest metabolic pathways. It has great relevance at a physiological level because it is responsible for generating ATP in the cell through the conversi...Glycolysis, present in most organisms, is evolutionarily one of the oldest metabolic pathways. It has great relevance at a physiological level because it is responsible for generating ATP in the cell through the conversion of glucose into pyruvate and reducing nicotinamide adenine dinucleotide (NADH) (that may be fed into the electron chain in the mitochondria to produce additional ATP by oxidative phosphorylation), as well as for producing intermediates that can serve as substrates for other metabolic processes. Glycolysis takes place through 10 consecutive chemical reactions, each of which is catalysed by a specific enzyme. Although energy transduction by glucose metabolism is the main function of this pathway, involvement in virulence, growth, pathogen-host interactions, immunomodulation and adaptation to environmental conditions are other functions attributed to this metabolic pathway. In humans, where glycolysis occurs mainly in the cytosol, the mislocalization of some glycolytic enzymes in various other subcellular locations, as well as alterations in their expression and regulation, has been associated with the development and progression of various diseases. In this review, we describe the role of glycolytic enzymes in the pathogenesis of diseases of clinical interest. In addition, the potential role of these enzymes as targets for drug development and their potential for use as diagnostic and prognostic markers of some pathologies are also discussed.
Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgeni...Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgenic models expressing fluorescent reporters for the selective autophagy of peroxisomes, a process known as pexophagy. We show that these reporters are colocalized with a peroxisomal marker and that they can reflect pexophagy induction by iron chelation and inhibition by depletion of the core autophagy protein Atg5. Using light sheet microscopy, we have been able to obtain a global overview of pexophagy levels across the entire organism at different stages of development. Tissue-specific control of pexophagy is exemplified by areas of peroxisome abundance but minimal pexophagy, observed in clusters of oenocytes surrounded by epithelial cells where pexophagy is much more evident. Enhancement of pexophagy was achieved by feeding flies with the iron chelator deferiprone, in line with past results using mammalian cells. Specific drivers were used to visualize pexophagy in neurons, and to demonstrate that specific depletion in the larval central nervous system of Hsc70-5, the homologue of the chaperone HSPA9/mortalin, led to a substantial elevation in pexophagy.
G-protein-coupled receptors (GPCRs) are efficient guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G-protein heterotrimers in response to various extracellular stimuli, including ne...G-protein-coupled receptors (GPCRs) are efficient guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G-protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP and free Gβγ and are major disease drivers. Evidence shows that the ambient low threshold signalling required for cells is likely supplemented by signalling regulators such as non-GPCR GEFs and guanine nucleotide dissociation inhibitors (GDIs). Activators of G-protein signalling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signalling regulation in living cells. Here, we employed structure-guided engineering of a novel optogenetic GDI, based on the AGS3's G-protein regulatory motif, to understand its GDI activity and induce standalone Gβγ signalling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gβγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signalling pathways and triggering GPCR-independent Gβγ signalling in cells and .
Epithelia are multicellular sheets that form barriers defining the internal and external environments. The constant stresses acting at this interface require that epithelial sheets are mechanically robust and provide a s...Epithelia are multicellular sheets that form barriers defining the internal and external environments. The constant stresses acting at this interface require that epithelial sheets are mechanically robust and provide a selective barrier to the hostile exterior. These properties are mediated by cellular junctions which are physically linked with heavily crosslinked cytoskeletal networks. Such hardwiring is facilitated by plakins, a family of giant modular proteins which serve as 'molecular bridges' between different cytoskeletal filaments and multiprotein adhesion complexes. Dysfunction of cytoskeletal crosslinking compromises epithelial biomechanics and structural integrity. Subsequent loss of barrier function leads to disturbed tissue homeostasis and pathological consequences such as skin blistering or intestinal inflammation. In this article, we highlight the importance of the cytolinker protein plectin for the functional organization of epithelial cytoskeletal networks. In particular, we focus on the ability of plectin to act as an integrator of the epithelial cytoarchitecture that defines the biomechanics of the whole tissue. Finally, we also discuss the role of cytoskeletal crosslinking in emerging aspects of epithelial mechanobiology that are critical for the maintenance of epithelial homeostasis.
Retrotransposon Gag-like (RTL) 8A, 8B and 8C are eutherian-specific genes derived from a certain retrovirus. They cluster as a triplet of genes on the X chromosome, but their function remains unknown. Here, we demonstrat...Retrotransposon Gag-like (RTL) 8A, 8B and 8C are eutherian-specific genes derived from a certain retrovirus. They cluster as a triplet of genes on the X chromosome, but their function remains unknown. Here, we demonstrate that and play important roles in the brain: their double knockout (DKO) mice not only exhibit reduced social responses and increased apathy-like behaviour, but also become obese from young adulthood, similar to patients with late Prader-Willi syndrome (PWS), a neurodevelopmental genomic imprinting disorder. Mouse RTL8A/8B proteins are expressed in the prefrontal cortex and hypothalamus and localize to both the nucleus and cytoplasm of neurons, presumably due to the N-terminal nuclear localization signal-like sequence at the N-terminus. An RNAseq study in the cerebral cortex revealed reduced expression of several GABA type A receptor subunit genes in DKO, in particular which encodes its β2 subunit. We confirmed the reduction of GABRB2 protein in the DKO cerebral cortex by western blotting. As GABRB2 has been implicated in the aetiology of several neurodevelopmental and neuropsychiatric disorders, it is likely that the reduction of GABRB2 is one of the major causes of the neuropsychiatric defects in the DKO mice.
Trace elements are often omitted from chemically defined growth media. From established properties of trace elements, we deduce that this omission makes experiments unnecessarily sensitive to unavoidable contamination wi...Trace elements are often omitted from chemically defined growth media. From established properties of trace elements, we deduce that this omission makes experiments unnecessarily sensitive to unavoidable contamination with trace elements. We confirm this experimentally by growing 11 bacterial strains in high replicate with and without supplementing trace elements, keeping all other conditions as fixed as possible to isolate the effect of trace elements. We find that supplementing trace elements considerably reduces variability of growth even in this benign scenario, and we argue that typical experimental set-ups exacerbate this. We discuss implications for the design and use of trace-element supplements and in particular argue that their use should be standard practice, as they can reduce variability of almost all experiments using chemically defined media, taking a step towards greater precision and replicability in microbiology.
The vertebrate visual cycle hinges on enzymatically converting all--retinol (at-ROL) into 11--retinal (11c-RAL), the chromophore that binds to opsins in photoreceptors, forming light-responsive pigments. When struck by a...The vertebrate visual cycle hinges on enzymatically converting all--retinol (at-ROL) into 11--retinal (11c-RAL), the chromophore that binds to opsins in photoreceptors, forming light-responsive pigments. When struck by a photon, these pigments activate the phototransduction pathway and initiate the process of vision. The enzymatic isomerization of at-ROL, crucial for restoring the visual pigments and preparing them to receive new light stimuli, relies on various enzymes found in both the photoreceptors and retinal pigment epithelium cells. To function effectively, retinoids must shuttle between these two cell types. Retinol-binding protein 3 (RBP3), located in the interphotoreceptor matrix, probably plays a pivotal role in this transport mechanism. Comprised of four retinoid-binding modules, RBP3 also binds fatty acids, potentially aiding retinal function by facilitating the loading and unloading of different retinoids at specific cell types thereby directing the cycle. In this study, we present a 3.67 Å cryoEM structure of porcine RBP3, along with molecular docking analysis and corroborative in-solution small-angle X-ray scattering data for titration of RBP3 with relevant ligands, that also give insights on RBP3 conformational adaptability.
Thrombin is generated from prothrombin through sequential cleavage at two sites by the enzyme complex prothrombinase, composed of a serine protease, factor (f) Xa and a cofactor, fVa, on phospholipid membranes. In a rece...Thrombin is generated from prothrombin through sequential cleavage at two sites by the enzyme complex prothrombinase, composed of a serine protease, factor (f) Xa and a cofactor, fVa, on phospholipid membranes. In a recent paper published in , Ruben . (Ruben . 2022 , 3463-3473 (doi:10.1182/blood.2022015807)) reported a major breakthrough in the field: the cryogenic electron microscopy structures of human prothrombinase on nanodiscs at 5.5 Å resolution (7TPQ) and of a catalytically inert human prothrombinase with its substrate prothrombin in the absence of any membrane at 4.1 Å resolution (7TPP). As is the norm in structural biology, the original paper was reviewed without access to the coordinates and maps, and it was therefore not possible for referees to assess the validity of the structures or their interpretations. In this article, we provide a post hoc analysis of the quality of the reported coordinates and maps, and look closely at the claimed intermolecular contacts on which the supposed breakthrough depends. We demonstrate that the work is deeply flawed, with not a single claimed intermolecular contact supported by the map, and conclude that the two reported structures do not contain any useful information regarding the assembly or function of the prothrombinase complex.
Sea urchins, integral to marine ecosystems and valued as a delicacy in Asia and Europe, contain physiologically important long-chain (>C) polyunsaturated fatty acids (PUFA) in their gonads, including arachidonic acid (AR...Sea urchins, integral to marine ecosystems and valued as a delicacy in Asia and Europe, contain physiologically important long-chain (>C) polyunsaturated fatty acids (PUFA) in their gonads, including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and unusual non-methylene-interrupted fatty acids (NMI-FA) such as 20:2. Although these fatty acids may partially be derived from their diet, such as macroalgae, the present study on has uncovered multiple genes encoding enzymes involved in long-chain PUFA biosynthesis. Specifically, 3 fatty acid desaturases (FadsA, FadsC1 and FadsC2) and 13 elongation of very-long-chain fatty acids proteins (Elovl-like, Elovl1/7-like, Elovl2/5-like, Elovl4-like, Elovl8-like and Elovl6-like A-H) were identified in their genome and transcriptomes. Functional analysis showed that FadsA and FadsC2 function as a Δ5 desaturase and a Δ8 desaturase, respectively, enabling the conversion of 18:2n-6 and 18:3n-3 into ARA and EPA, respectively, along with Elovl, particularly Elovl6-like C. Elovl6-like C demonstrates elongase activity towards both C PUFA and monounsaturated fatty acids. Consequently, FadsA and Elovl6-like C enable the synthesis of several NMI-FA, including 20:2 and 20:3, from C precursors. This indicates that can endogenously synthesize a wide variety of C PUFA and NMI-FA, highlighting active biosynthesis pathways within sea urchins.