Publications - Publikationer

Improving laboratory animal genetic reporting

Teboul, L., Amos-Landgraf, J., Benavides, F. J., et al. — Mon, 01 Jul 2024 05:08:20 +0200

The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework. LAG-R aims to document animals' genetic makeup in scientific publications, providing essential details for replication and appropriate model use. While verifying complete genetic compositions may be impractical, better reporting and validation efforts enhance reliability of research. LAG-R standardization will bolster reproducibility, peer review, and overall scientific rigor.

A suicidal and extensively disordered luciferase with a bright luminescence

Dijkema, F. M., Escarpizo-Lorenzana, M. I., Nordentoft, M. K., et al. — Thu, 01 Aug 2024 05:08:20 +0200

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

Integrative network analysis of miRNA-mRNA expression profiles during epileptogenesis in rats reveals therapeutic targets after emergence of first spontaneous seizure

Khemka, N., Morris, G., Kazemzadeh, L., et al. — Sun, 01 Dec 2024 05:08:20 +0100

Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) – short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS

Adam, L., Kumar, R., Arroyo-Garcia, L. E., et al. — Thu, 01 Aug 2024 05:08:20 +0200

Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

Assigning function to an unexplored Integrator module

Garland, W., Rouvière, J. O., Heick Jensen, T. — Thu, 11 Jul 2024 05:08:20 +0200

In this issue of Molecular Cell, Razew et al.¹ and Sabath et al.² assign function to an unexplored module of the Integrator (INT) complex, expanding the toolbox of this genome-wide attenuator of RNA polymerase II (RNAPII) transcription.

Mapping the Periostin splice isoforms in atopic dermatitis and an in vitro asthma model - A multi-platform analysis using mass spectrometry and RT-qPCR

Rusbjerg-Weberskov, C. E., Hollensen, A. K., Damgaard, C. K., et al. — Sun, 01 Sep 2024 05:08:20 +0200

Periostin is a matricellular protein known to be alternatively spliced to produce ten isoforms with a molecular weight of 78-91 kDa. Within the extracellular matrix, periostin attaches to cell surfaces to induce signaling via integrin-binding and actively participates in fibrillogenesis, orchestrating the arrangement of collagen in the extracellular environment. In atopic diseases such as atopic dermatitis (AD) and asthma, periostin is known to participate in driving the disease-causing type 2 inflammation. The periostin isoforms expressed in these diseases and the implication of the alternative splicing events are unknown. Here, we present two universal assays to map the expression of periostin isoforms at the mRNA (RT-qPCR) and protein (PRM-based mass spectrometry) levels. We use these assays to study the splicing profile of periostin in AD lesions as well as in in vitro models of AD and asthma. In these conditions, periostin displayed overexpression with isoforms 3 and 5 standing out as highly overexpressed. Notably, isoforms 9 and 10 exhibited a divergent pattern relative to the remaining isoforms. Isoforms 9 and 10 are often overlooked in periostin research and this paper presents the first evidence of their expression at the protein level. This underlines the necessity to include isoforms 9 and 10 in future research addressing periostin splice isoforms. The assays presented in this paper hold the potential to improve our insight into the splicing profile of periostin in tissues and diseases of interest. The application of these assays to AD lesions and in vitro models demonstrated their potential for identifying isoforms of particular significance, warranting a further in-depth investigation.

Development of a top-down MS assay for specific identification of human periostin isoforms

Mon, 01 Jan 2024 05:08:20 +0100

Periostin is a matricellular protein encoded by the POSTN gene that is alternatively spliced to produce ten different periostin isoforms with molecular weights ranging from 78 to 91 kDa. It is known to promote fibrillogenesis, organize the extracellular matrix, and bind integrin-receptors to induce cell signaling. As well as being a key component of the wound healing process, it is also known to participate in the pathogenesis of different diseases including atopic dermatitis, asthma, and cancer. In both health and disease, the functions of the different periostin isoforms are largely unknown. The ability to precisely determine the isoform profile of a given human sample is fundamental for characterizing their functional significance. Identification of periostin isoforms is most often carried out at the transcriptional level using RT-PCR based approaches, but due to high sequence homogeneity, identification on the protein level has always been challenging. Top-down proteomics, where whole proteins are measured by mass spectrometry, offers a fast and reliable method for isoform identification. Here we present a fully developed top-down mass spectrometry assay for the characterization of periostin splice isoforms at the protein level.

Characterization of a Trispecific PD-L1 Blocking Antibody That Exhibits EGFR-Conditional 4-1BB Agonist Activity

Rubio-Pérez, L., Frago, S., Compte, M., et al. — Sat, 01 Jun 2024 05:08:20 +0200

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further clinical development of these antibodies has been hampered by significant off-tumor toxicities. Here, we generated an anti-4-1BB/EGFR/PD-L1 trispecific antibody consisting of a triple-targeting tandem trimerbody (TT) fused to an engineered silent Fc region. This antibody (IgTT-4E1-S) was designed to combine the blockade of the PD-L1/PD-1 axis with conditional 4-1BB costimulation specifically confined to the tumor microenvironment (TME). The antibody demonstrated simultaneous binding to purified EGFR, PD-L1, and 4-1BB in solution, effective blockade of the PD-L1/PD1 interaction, and potent 4-1BB-mediated costimulation, but only in the presence of EGFR-expressing cells. These results demonstrate the feasibility of IgTT-4E1-S specifically blocking the PD-L1/PD-1 axis and inducing EGFR-conditional 4-1BB agonist activity.

Unbiased plasma profiling using pre-selected RNA aptamer pools predicts mortality in COVID-19 and identifies protein risk factors

Jørgensen, A. G., Dupont, D. M., Fjelstrup, S., et al. — Sun, 01 Sep 2024 05:08:20 +0200

The impact of the COVID-19 pandemic demands effective prognostic tools for precise risk evaluation and timely intervention. This study utilized the APTASHAPE technology to profile plasma proteins in COVID-19 patient samples. Employing a highly diverse 2′-fluoro-protected RNA aptamer pool enriched toward proteins in the plasma samples from COVID-19 patients, we performed a single round of parallel selection on the derivation cohort and identified 93 discriminatory aptamers capable of distinguishing COVID-19 and healthy plasma samples. A subset of these aptamers was then used to predict 30-day mortality with high sensitivity and specificity in a validation cohort of 165 patients. We predicted 30-day mortality with areas under the curve (AUCs) of 0.91 in females and 0.68 in males. Affinity purification coupled with mass spectrometry analysis of the aptamer-targeted proteins identified potential biomarkers associated with disease severity, including complement system components. The study demonstrates the APTASHAPE technology as an unbiased approach that not only aids in predicting disease outcomes but also offers insights into gender-specific differences, shedding light on the nuanced aspects of COVID-19 pathophysiology. In conclusion, the findings highlight the promise of APTASHAPE as a valuable tool for estimating risk factors in COVID-19 patients and enabling stratification for personalized treatment management.

Compositionally aware estimation of crosscorrelations for microbiome data

Jensen, I. T., Janss, L., Radutoiu, S., Waagepetersen, R. — Sat, 01 Jun 2024 05:08:20 +0200

In the field of microbiome studies, it is of interest to infer correlations between abundances of different microbes (here referred to as operational taxonomic units, OTUs). Several methods taking the compositional nature of the sequencing data into account exist. However, these methods cannot infer correlations between OTU abundances and other variables. In this paper we introduce the novel methods SparCEV (Sparse Correlations with External Variables) and SparXCC (Sparse Cross-Correlations between Compositional data) for quantifying correlations between OTU abundances and either continuous phenotypic variables or components of other compositional datasets, such as transcriptomic data. Spar- CEV and SparXCC both assume that the average correlation in the dataset is zero. Iterative versions of SparCEV and SparXCC are proposed to alleviate bias resulting from deviations from this assumption. We compare these new methods to empirical Pearson cross-correlations after applying naive transformations of the data (log and log-TSS). Additionally, we test the centered log ratio transformation (CLR) and the variance stabilising transformation (VST). We find that CLR and VST outperform naive transformations, except when the correlation matrix is dense. SparCEV and SparXCC outperform CLR and VST when the number of OTUs is small and perform similarly to CLR and VST for large numbers of OTUs. Adding the iterative procedure increases accuracy for SparCEV and SparXCC for all cases, except when the average correlation in the dataset is close to zero or the correlation matrix is dense. These results are consistent with our theoretical considerations.

Phenotypic and ancestry-related assortative mating in autism

Zhang, J., Weissenkampen, J. D., Kember, R. L., et al. — Sat, 01 Jun 2024 05:08:20 +0200

BACKGROUND: Positive assortative mating (AM) in several neuropsychiatric traits, including autism, has been noted. However, it is unknown whether the pattern of AM is different in phenotypically defined autism subgroups [e.g., autism with and without intellectually disability (ID)]. It is also unclear what proportion of the phenotypic AM can be explained by the genetic similarity between parents of children with an autism diagnosis, and the consequences of AM on the genetic structure of the population. METHODS: To address these questions, we analyzed two family-based autism collections: the Simons Foundation Powering Autism Research for Knowledge (SPARK) (1575 families) and the Simons Simplex Collection (SSC) (2283 families). RESULTS: We found a similar degree of phenotypic and ancestry-related AM in parents of children with an autism diagnosis regardless of the presence of ID. We did not find evidence of AM for autism based on autism polygenic scores (PGS) (at a threshold of |r|> 0.1). The adjustment of ancestry-related AM or autism PGS accounted for only 0.3-4% of the fractional change in the estimate of the phenotypic AM. The ancestry-related AM introduced higher long-range linkage disequilibrium (LD) between single nucleotide polymorphisms (SNPs) on different chromosomes that are highly ancestry-informative compared to SNPs that are less ancestry-informative (D2 on the order of 1 × 10-5). LIMITATIONS: We only analyzed participants of European ancestry, limiting the generalizability of our results to individuals of non-European ancestry. SPARK and SSC were both multicenter studies. Therefore, there could be ancestry-related AM in SPARK and SSC due to geographic stratification. The study participants from each site were unknown, so we were unable to evaluate for geographic stratification. CONCLUSIONS: This study showed similar patterns of AM in autism with and without ID, and demonstrated that the common genetic influences of autism are likely relevant to both autism groups. The adjustment of ancestry-related AM and autism PGS accounted for < 5% of the fractional change in the estimate of the phenotypic AM. Future studies are needed to evaluate if the small increase of long-range LD induced by ancestry-related AM has impact on the downstream analysis.

Alternative electron sinks in chloroplasts and mitochondria of halophytes as a safety valve for controlling ROS production during salinity

Demircan, N., Sonmez, M. C., Akyol, T. Y., et al. — Wed, 01 May 2024 05:08:20 +0200

Electron flow through the electron transport chain (ETC) is essential for oxidative phosphorylation in mitochondria and photosynthesis in chloroplasts. Electron fluxes depend on environmental parameters, e.g., ionic and osmotic conditions and endogenous factors, and this may cause severe imbalances. Plants have evolved alternative sinks to balance the reductive load on the electron transport chains in order to avoid overreduction, generation of reactive oxygen species (ROS), and to cope with environmental stresses. These sinks act primarily as valves for electron drainage and secondarily as regulators of tolerance-related metabolism, utilizing the excess reductive energy. High salinity is an environmental stressor that stimulates the generation of ROS and oxidative stress, which affects growth and development by disrupting the redox homeostasis of plants. While glycophytic plants are sensitive to high salinity, halophytic plants tolerate, grow, and reproduce at high salinity. Various studies have examined the ETC systems of glycophytic plants, however, information about the state and regulation of ETCs in halophytes under non-saline and saline conditions is scarce. This review focuses on alternative electron sinks in chloroplasts and mitochondria of halophytic plants. In cases where information on halophytes is lacking, we examined the available knowledge on the relationship between alternative sinks and gradual salinity resilience of glycophytes. To this end, transcriptional responses of involved components of photosynthetic and respiratory ETCs were compared between the glycophyte Arabidopsis thaliana and the halophyte Schrenkiella parvula, and the time-courses of these transcripts were examined in A. thaliana. The observed regulatory patterns are discussed in the context of reactive molecular species formation in halophytes and glycophytes.

An expert panel on the adequacy of safety data and physiological roles of dietary bovine osteopontin in infancy

Fleming, S. A., Reyes, S. M., Donovan, S. M., et al. — Mon, 01 Jan 2024 05:08:20 +0100

Human milk, due to its unique composition, is the optimal standard for infant nutrition. Osteopontin (OPN) is abundant in human milk but not bovine milk. The addition of bovine milk osteopontin (bmOPN) to formula may replicate OPN’s concentration and function in human milk. To address safety concerns, we convened an expert panel to assess the adequacy of safety data and physiological roles of dietary bmOPN in infancy. The exposure of breastfed infants to human milk OPN (hmOPN) has been well-characterized and decreases markedly over the first 6 months of lactation. Dietary bmOPN is resistant to gastric and intestinal digestion, absorbed and cleared from circulation within 8–24 h, and represents a small portion (<5%) of total plasma OPN. Label studies on hmOPN suggest that after 3 h, intact or digested OPN is absorbed into carcass (62%), small intestine (23%), stomach (5%), and small intestinal perfusate (4%), with <2% each found in the cecum, liver, brain, heart, and spleen. Although the results are heterogenous with respect to bmOPN’s physiologic impact, no adverse impacts have been reported across growth, gastrointestinal, immune, or brain-related outcomes. Recombinant bovine and human forms demonstrate similar absorption in plasma as bmOPN, as well as effects on cognition and immunity. The panel recommended prioritization of trials measuring a comprehensive set of clinically relevant outcomes on immunity and cognition to confirm the safety of bmOPN over that of further research on its absorption, distribution, metabolism, and excretion. This review offers expert consensus on the adequacy of data available to assess the safety of bmOPN for use in infant formula, aiding evidence-based decisions on the formulation of infant formula.

RNA nanostructures for targeted drug delivery and imaging

Teodori, L., Omer, M., Kjems, J. — Sun, 31 Mar 2024 05:08:20 +0200

The RNA molecule plays a pivotal role in many biological processes by relaying genetic information, regulating gene expression, and serving as molecular machines and catalyzers. This inherent versatility of RNA has fueled significant advancements in the field of RNA nanotechnology, driving the engineering of complex nanoscale architectures toward biomedical applications, including targeted drug delivery and bioimaging. RNA polymers, serving as building blocks, offer programmability and predictability of Watson-Crick base pairing, as well as non-canonical base pairing, for the construction of nanostructures with high precision and stoichiometry. Leveraging the ease of chemical modifications to protect the RNA from degradation, researchers have developed highly functional and biocompatible RNA architectures and integrated them into preclinical studies for the delivery of payloads and imaging agents. This review offers an educational introduction to the use of RNA as a biopolymer in the design of multifunctional nanostructures applied to targeted delivery in vivo, summarizing physical and biological barriers along with strategies to overcome them. Furthermore, we highlight the most recent progress in the development of both small and larger RNA nanostructures, with a particular focus on imaging reagents and targeted cancer therapeutics in pre-clinical models and provide insights into the prospects of this rapidly evolving field.

Assessing metabolic rates in zebrafish using a 3D-printed intermittent-flow respirometer and swim tunnel system

Hejlesen, R., Scheffler, F. B., Byrge, C. G., et al. — Sat, 01 Jun 2024 05:08:20 +0200

Zebrafish have become a widely used vertebrate model in physiology
and reliable measures of their metabolic rate are needed. We have
developed a 3D-printed respirometer and swim tunnel system and
used it for obtaining accurate measurement of standard metabolic
rate (SMR) and maximal, aerobic metabolic rate (MMR) in zebrafish
under rest and maximal exercise, respectively. We compared a slow
(stepwise) protocol to a fast (continuous) protocol for determining
MMR. The fast protocol yielded slightly (but not significantly) higher
oxygen consumption rates than the slow protocol and the data, in
contrast to the slow protocol, followed a normal distribution. These
findings point to the fast protocol as a fast and reliable method for
obtaining accurate values of MMR in zebrafish. We make the 3D
drawings for printing the system available to researchers, to help
streamline the field of metabolic research in zebrafish and other
smaller fish species.

Zinc mediates control of nitrogen fixation via transcription factor filamentation

Lin, J., Bjørk, P. K., Kolte, M. V., et al. — Thu, 04 Jul 2024 05:08:20 +0200

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness 1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules 2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Phenotyping revealed tolerance traits and genotypes for acidity and aluminum toxicity in European Vicia faba L

Belachew, K. Y., Skovbjerg, C. K., Andersen, S. U., Stoddard, F. L. — Wed, 01 May 2024 05:08:20 +0200

Soil acidity is a global issue; soils with pH <4.5 are widespread in Europe. This acidity adversely affects nutrient availability to plants; pH levels <5.0 lead to aluminum (Al³⁺) toxicity, a significant problem that hinders root growth and nutrient uptake in faba bean (Vicia faba L.) and its symbiotic relationship with Rhizobium. However, little is known about the specific traits and tolerant genotypes among the European faba beans. This study aimed to identify response traits associated with tolerance to root zone acidity and Al³⁺ toxicity and potentially tolerant genotypes for future breeding efforts. Germplasm survey was conducted using 165 genotypes in a greenhouse aquaponics system. Data on the root and shoot systems were collected. Subsequently, 12 genotypes were selected for further phenotyping in peat medium, where data on physiological and morphological parameters were recorded along with biochemical responses in four selected genotypes. In the germplasm survey, about 30% of genotypes showed tolerance to acidity and approximately 10% exhibited tolerance to Al³⁺, while 7% showed tolerance to both. The phenotyping experiment indicated diverse morphological and physiological responses among treatments and genotypes. Acid and Al³⁺ increased proline concentration. Interaction between genotype and environment was observed for ascorbate peroxidase activity, malondialdehyde, and proline concentrations. Genomic markers associated with acidity and acid+Al³⁺-toxicity tolerances were identified using GWAS analysis. Four faba bean genotypes with varying levels of tolerance to acidity and Al³⁺ toxicity were identified.

Amperometric biosensor modified with platinum and palladium nanoparticles for detection of lactate concentrations in wine

Shkotova, L., Bohush, A., Voloshina, I., Smutok, O., Dzyadevych, S. — Mon, 01 Apr 2019 05:08:20 +0200

Detection of the lactate content is of great importance in clinical diagnostics, fermentation industry, and control of the quality of food products. The work was aimed at the development of a sensitive element of the amperometric biosensor, based on enzyme lactate oxidase and carbon electrodes modified with platinum and palladium nanoparticles (Pt&Pd), for the lactate analysis in the presence of interfering substances. The voltamperometric characteristics of the modified sensor were studied, the enzyme stabilization was carried out. An influence of the medium parameters on the biosensor operation was comprehensively investigated. The working characteristics of the biosensor were thoroughly analyzed, its stability and selectivity were investigated. An increase in the bioselective membrane activity as a result of using Pt&Pd nanoparticles was shown. The developed biosensor for the measurement of lactate concentration is characterized by the linear range of 0.05–0.8 mM, the lower detection limit 0.1 µM and sensitivity of 3.03 nA mM⁻¹ cm⁻². The main interferents were shown to have no effect on the work of created lactate biosensor. The lactate content in several types of wine and must was determined with created biosensor (lactate concentration in wine ranges from 0.5 to 5 g/l), and the results were compared with those obtained by the traditional spectrophotometric method; good correlation was shown (the correlation coefficient R² = 0.98). The developed sensor can be used in winemaking for selective detection of lactate in raw material during fermentation and control of the final quality of wine.

Hsp90 and Its Co-Chaperones in Neurodegenerative Diseases

Bohush, A., Bieganowski, P., Filipek, A. — Wed, 09 Oct 2019 05:08:20 +0200

Proper folding is crucial for proteins to achieve functional activity in the cell. However, it often occurs that proteins are improperly folded (misfolded) and form aggregates, which are the main hallmark of many diseases including cancers, neurodegenerative diseases and many others. Proteins that assist other proteins in proper folding into three-dimensional structures are chaperones and co-chaperones. The key role of chaperones/co-chaperones is to prevent protein aggregation, especially under stress. An imbalance between chaperone/co-chaperone levels has been documented in neurons, and suggested to contribute to protein misfolding. An essential protein and a major regulator of protein folding in all eukaryotic cells is the heat shock protein 90 (Hsp90). The function of Hsp90 is tightly regulated by many factors, including co-chaperones. In this review we summarize results regarding the role of Hsp90 and its co-chaperones in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and prionopathies.

Calmodulin and Its Binding Proteins in Parkinson's Disease

Bohush, A., Leśniak, W., Weis, S., Filipek, A. — Tue, 16 Mar 2021 05:08:20 +0100

Parkinson's disease (PD) is a neurodegenerative disorder that manifests with rest tremor, muscle rigidity and movement disturbances. At the microscopic level it is characterized by formation of specific intraneuronal inclusions, called Lewy bodies (LBs), and by a progressive loss of dopaminergic neurons in the striatum and substantia nigra. All living cells, among them neurons, rely on Ca2+ as a universal carrier of extracellular and intracellular signals that can initiate and control various cellular processes. Disturbances in Ca2+ homeostasis and dysfunction of Ca2+ signaling pathways may have serious consequences on cells and even result in cell death. Dopaminergic neurons are particularly sensitive to any changes in intracellular Ca2+ level. The best known and studied Ca2+ sensor in eukaryotic cells is calmodulin. Calmodulin binds Ca2+ with high affinity and regulates the activity of a plethora of proteins. In the brain, calmodulin and its binding proteins play a crucial role in regulation of the activity of synaptic proteins and in the maintenance of neuronal plasticity. Thus, any changes in activity of these proteins might be linked to the development and progression of neurodegenerative disorders including PD. This review aims to summarize published results regarding the role of calmodulin and its binding proteins in pathology and pathogenesis of PD.

Role of Mitogen Activated Protein Kinase Signaling in Parkinson's Disease

Bohush, A., Niewiadomska, G., Filipek, A. — Sat, 29 Sep 2018 05:08:20 +0200

Parkinson's disease (PD) is a neurodegenerative disorder caused by insufficient dopamine production due to the loss of 50% to 70% of dopaminergic neurons. A shortage of dopamine, which is predominantly produced by the dopaminergic neurons within the substantia nigra, causes clinical symptoms such as reduction of muscle mass, impaired body balance, akinesia, bradykinesia, tremors, postural instability, etc. Lastly, this can lead to a total loss of physical movement and death. Since no cure for PD has been developed up to now, researchers using cell cultures and animal models focus their work on searching for potential therapeutic targets in order to develop effective treatments. In recent years, genetic studies have prominently advocated for the role of improper protein phosphorylation caused by a dysfunction in kinases and/or phosphatases as an important player in progression and pathogenesis of PD. Thus, in this review, we focus on the role of selected MAP kinases such as JNKs, ERK1/2, and p38 MAP kinases in PD pathology.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes

Leśniak, W., Bohush, A., Maksymowicz, M., et al. — Mon, 01 May 2023 05:08:20 +0200

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

Sgt1 Regulates α-Synuclein Subcellular Localization and Expression of Parkinson's Disease Related Genes, PINK1 and PARK9

Bohush, A., Góral, A., Sierant, M., Nawrot, B., Leśniak, W., Filipek, A. — Thu, 11 Nov 2021 05:08:20 +0100

The SGT1 protein is highly expressed in the mammalian brain, particularly in neurons of the hippocampus and cortex, and in Purkinje cells of the cerebellum. There are literature data indicating that the protein may be involved in pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). In the present work we have found that SGT1 protected cells from the toxicity of rotenone, an agent that evokes behavioral and histopathological symptoms of PD. To gain more insight into the possible mechanism underlying the protective action of SGT1 we looked at α-synuclein subcellular distribution in HEK293 cells with an altered SGT1 level. By immunofluorescent staining we have found that in HEK293 cells overexpressing SGT1 α-synuclein was mainly localized in the cytoplasm while in control cells it was present in the nucleus. Accordingly, when SGT1 expression was silenced, α-synuclein was predominantly present in the nucleus. These results were then confirmed by subcellular fractionation and Western blot analysis. Moreover, we have found that altered level of SGT1 in HEK293 cells influenced the expression of PD related genes, PINK1 and PARK9. Altogether, our results point to SGT1 as an important factor that might be involved in the pathogenesis of Parkinson's disease (PD).

HSP90 and Its Novel Co-Chaperones, SGT1 and CHP-1, in Brain of Patients with Parkinson's Disease and Dementia with Lewy Bodies

Bohush, A., Niewiadomska, G., Weis, S., Filipek, A. — Tue, 01 Jan 2019 05:08:20 +0100

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of inclusions known as Lewy bodies in some brain regions. Lewy bodies consist of α-synuclein and many other proteins including chaperones.

OBJECTIVE: To learn more about the role of chaperone complexes in PD and a related disorder, i.e., dementia with Lewy bodies (DLB), in this work we analyzed the expression of HSP90 and its two quite recently identified co-chaperones, SGT1 and CHP-1, in selected brain regions from patients suffering from these diseases.

METHODS: To fulfill the aim of our study we used human material and applied immunohistochemistry, Western blot analysis and real time/quantitative PCR (RT-qPCR).

RESULTS: We have found that HSP90 mRNA level is higher in the temporal cortex of PD and in frontal cortex of DLB brains, even though level of protein does not change significantly. The mRNA level of SGT1 is higher in the frontal and temporal cortex of PD and in substantia nigra of DLB brains while no significant changes in the level of protein were noticed. Similarly, the mRNA level of CHP-1 was found to be higher in the frontal and temporal cortex of PD and in all examined regions i.e. substantia nigra, frontal and temporal cortex of DLB brains. In the case of CHP-1 the protein level was found to be higher in frontal cortex of PD and in all examined areas of DLB patients.

CONCLUSIONS: Our data indicate that the level of HSP90, SGT1 and CHP-1 is upregulated in the majority of cases of PD and DLB, which suggests that the examined proteins might be involved in these pathologies.

HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

Bohush, A., Filipek, A. — Thu, 08 Oct 2020 05:08:20 +0200

Recently, it has been found that the CacyBP/SIP protein acts as HSP90 co-chaperone and exhibits chaperone properties itself. Namely, CacyBP/SIP has been shown to protect citrate synthase from aggregation and to recover the activity of thermally denatured luciferase in vitro. In the present work, we have analyzed the influence of CacyBP/SIP on aggregation of α-synuclein, a protein present in Lewy bodies of Parkinson's disease brain. By applying a thioflavin T (ThT) fluorescence assay, we have found that CacyBP/SIP protects α-synuclein from aggregation and that the fragment overlapping the N-terminal part and the CS domain of CacyBP/SIP is crucial for this activity. This protective effect of CacyBP/SIP has been confirmed by results obtained using high-speed ultracentrifugation followed by dot-blot and by transmission electron microscopy (TEM). Interestingly, CacyBP/SIP exhibits the protective effect only at the initial phase of α-synuclein aggregation. In addition, we have found that, in HEK293 cells overexpressing CacyBP/SIP, there are less α-synuclein inclusions than in control ones. Moreover, these cells are more viable when treated with rotenone, an agent that mimics PD pathology. By applying proximity ligation assay (PLA) on HEK293 cells and in vitro assays with the use of purified recombinant proteins, we have found that CacyBP/SIP directly interacts with α-synuclein. Altogether, in this work, we show for the first time that CacyBP/SIP is able to protect α-synuclein from aggregation in in vitro assays. Thus, our results point to an important role of CacyBP/SIP in the pathology of Parkinson's disease and other synucleinopathies.

Recent Advances on Catenanes and Rotaxanes Made of DNA

Ma, Y., Yu, Z., Valero, J. — Mon, 01 Jan 2024 05:08:20 +0100

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2

Rezi, C. K., Aslanyan, M. G., Diwan, G. D., et al. — Mon, 01 Jul 2024 05:08:20 +0200

(Figure presented.) The Scribble polarity complex protein DLG1 regulates ciliary length and protein composition in kidney epithelial cells. A Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant may be associated with ciliary dysfunction. Loss of DLG1 causes ciliary elongation in kidney epithelial cells. Loss of DLG1 impairs targeting of SDCCAG3, IFT20 and PC2 to the primary cilium of kidney epithelial cells. The CAKUT-associated p.T489R DLG1 fails to rescue ciliary defects of Dlg1^-/- cells, indicating a possible ciliary involvement in CAKUT disease etiology.

Differential regulation of the interferon induced gene ISG12A by serum from healthy and preeclamptic pregnancies

Kronborg, C. S., Knudsen, U. B., Martensen, P. M. — Tue, 01 Apr 2008 05:08:20 +0200

Endothelial-cell dysfunction is central in the preeclamptic pathogenesis. Several components present in the blood of the preeclamptic mother are capable of mediating this dysfunction. We analyzed the regulation of the ISG12A gene by serum from the third trimester, to elucidate the role of type 1 interferon ISGs late in both healthy and preeclamptic pregnancies. The ISG12A transcription was up-regulated by serum from healthy pregnant women, but not by preeclamptic serum in HeLa and human umbilical vein endothelial cells (HUVEC). However, the ISG12A up-regulation by healthy pregnancy serum was not due to a general type 1 interferon response, since 6-16 and OAS1 were not up-regulated similarly. Also, the up-regulation of ISG12A was independent of the interferon-α receptor 2, but dependent on STAT1. Stimulation with folic acid alone or in combination with preeclamptic serum up-regulated ISG12A and 6-16. We conclude that type 1 interferon is not increased in third trimester serum, neither from healthy nor preeclamptic pregnancies. However, since ISG12A mRNA is up-regulated in healthy pregnancies, the ISG12A protein might take part in maintaining endothelial stability, as this function is lacking in preeclamptic pregnancies. Folic acid may ameliorate endothelial cell stability in preeclampsia by up-regulating ISG12A.

Folic acid mediates activation of the pro-oncogene STAT3 via the Folate Receptor alpha

Hansen, M. F., Greibe, E., Skovbjerg, S., et al. — Wed, 01 Jul 2015 05:08:20 +0200

The signal transducer and activator of transcription 3 (STAT3) is a well-described pro-oncogene found constitutively activated in several cancer types. Folates are B vitamins that, when taken up by cells through the Reduced Folate Carrier (RFC), are essential for normal cell growth and replication. Many cancer cells overexpress a glycophosphatidylinositol (GPI)-anchored Folate Receptor α (FRα). The function of FRα in cancer cells is still poorly described, and it has been suggested that transport of folate is not its primary function in these cells.We show here that folic acid and folinic acid can activate STAT3 through FRα in a Janus Kinase (JAK)-dependent manner, and we demonstrate that gp130 functions as a transducing receptor for this signalling. Moreover, folic acid can promote dose dependent cell proliferation in FRα-positive HeLa cells, but not in FRα-negative HEK293 cells. After folic acid treatment of HeLa cells, up-regulation of the STAT3 responsive genes Cyclin A2 and Vascular Endothelial Growth Factor (VEGF) were verified by qRT-PCR.The identification of this FRα-STAT3 signal transduction pathway activated by folic and folinic acid contributes to the understanding of the involvement of folic acid in preventing neural tube defects as well as in tumour growth. Previously, the role of folates in these diseases has been attributed to their roles as one-carbon unit donors following endocytosis into the cell. Our finding that folic acid can activate STAT3 via FRα adds complexity to the established roles of B9 vitamins in cancer and neural tube defects.

A robust immunoassay for pregnancy-associated plasma protein-A2 based on analysis of circulating antigen

Kloverpris, S., Gaidamauskas, E., Rasmussen, L. C.V., et al. — Fri, 01 Nov 2013 05:08:20 +0100

Pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, two homologous metzincin metalloproteases, are both tightly linked to regulation within the insulin-like growth factor (IGF) system because of their specific cleavage of IGF binding proteins. Recent studies suggest that PAPP-A may be involved in clinical conditions related to unwanted cellular growth, and the circulating levels of PAPP-A is an established biomarker in prenatal screening for chromosomal abnormalities. Microarray data indicate that PAPP-A2 has potential as a biomarker for pre-eclampsia. However, well-characterized immunological methods of quantification are not available.We therefore developed monoclonal antibodies against recombinant PAPP-A2. The antibodies were epitope mapped against recombinantly expressed chimeras between PAPPA2 and PAPP-A. Furthermore, circulating PAPP-A2 was immunoaffinity purified and characterized by sequence analysis and mass spectrometry. Unlike PAPP-A, PAPP-A2 is a noncovalent dimer in which each subunit of 1558 amino acids originates fromall of the 22 predicted coding exons. A previously hypothesized variant (PAPP-E) does not exist, but low amounts of a C-terminally truncated PAPP-A2 variant was detected. A sensitive and robust ELISA for full-length PAPP-A2 was developed and used to establish normal ranges of PAPP-A2 through pregnancy.The functional sensitivity of this ELISA at 20% CV was 0.08 ng/ml, and the serum concentration of PAPP-A2 was found to increase during pregnancy in agreement with placental synthesis. The existence of this assay will enable an assessment of the biomarker potential of PAPP-A2 in pre-eclampsia as well as other clinical conditions.

Differential regulation of the interferon induced gene ISG12A by serum from healthy and preeclamptic pregnancies

Kronborg, C. S., Knudsen, U. B., Martensen, P. M. — Tue, 01 Apr 2008 05:08:20 +0200

Endothelial-cell dysfunction is central in the preeclamptic pathogenesis. Several components present in the blood of the preeclamptic mother are capable of mediating this dysfunction. We analyzed the regulation of the ISG12A gene by serum from the third trimester, to elucidate the role of type 1 interferon ISGs late in both healthy and preeclamptic pregnancies. The ISG12A transcription was up-regulated by serum from healthy pregnant women, but not by preeclamptic serum in HeLa and human umbilical vein endothelial cells (HUVEC). However, the ISG12A up-regulation by healthy pregnancy serum was not due to a general type 1 interferon response, since 6-16 and OAS1 were not up-regulated similarly. Also, the up-regulation of ISG12A was independent of the interferon-alpha receptor 2, but dependent on STAT1. Stimulation with folic acid alone or in combination with preeclamptic serum up-regulated ISG12A and 6-16. We conclude that type 1 interferon is not increased in third trimester serum, neither from healthy nor preeclamptic pregnancies. However, since ISG12A mRNA is up-regulated in healthy pregnancies, the ISG12A protein might take part in maintaining endothelial stability, as this function is lacking in preeclamptic pregnancies. Folic acid may ameliorate endothelial cell stability in preeclampsia by up-regulating ISG12A.

Reduction of serum-induced endothelial STAT3(Y705) activation is associated with preeclampsia

Sun, 01 Aug 2021 05:08:20 +0200

OBJECTIVES: Preeclampsia is associated with maternal morbidity and mortality during pregnancy, and also an increased cardiovascular disease (CVD) risk later in life. During preeclampsia, alterations in secreted placental factors leading to systemic maternal endothelial dysfunction are evident. However, little is known about the associated endothelial intracellular signaling. STAT3 is a latent cytoplasmic transcription factor involved in endothelial cell differentiation, survival, and angiogenesis. We aimed to test if preeclampsia and preeclampsia-related placental factors could alter serum-induced STAT3(Y705) activation in endothelial cells. Furthermore, if altered serum-induced endothelial STAT3 (Y705) activation is related to post-preeclamptic CVD risk.

STUDY DESIGN: HUVECs were used as a model of maternal endothelium. Experiments entailed addition of 20% human pregnancy serum as well as addition of recombinant PlGF, sFLT1 and VEGF-A165a to the cells.

MAIN OUTCOME MEASURES: Levels of pSTAT3(Y705) related to STAT3 levels were evaluated by immunoblotting analysis.

RESULTS: Our results show that preeclamptic serum induces significantly lower STAT3(Y705) phosphorylation compared with uncomplicated pregnancy serum (P = 0.0089) in endothelial cells. Furthermore, STAT3(Y705) phosphorylation was not changed upon addition of PlGF, sFLT1, or VEGF-A165a together with pregnancy sera compared with sera alone. Finally, sera from women with previous preeclampsia and current hypertension and carotid atherosclerotic plaques show significantly lower STAT3(Y705) phosphorylation capabilities compared with healthy women with previous uncomplicated pregnancies 8-18 years after deliveries (P = 0.029).

CONCLUSIONS: Reduction in serum-induced endothelial STAT3(Y705) activation may play an important role in the preeclampsia-associated endothelial dysfunction. Additionally, reduced endothelial STAT3(Y705) phosphorylation may contribute to increased post-preeclamptic CVD risk 8-18 years after delivery.

Dietary intake of a MFGM/EV-rich concentrate promotes accretion of very long odd-chain sphingolipids and increases lipid metabolic turnover at the whole-body level

Thu, 01 Aug 2024 05:08:20 +0200

Lipids from cow milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for neurodevelopment, cognitive maintenance and human health in general. Nevertheless, it is largely unknown whether intake of infant formulas and medical nutrition products rich in these particles promote accretion of specific lipids and whether this affects metabolic homeostasis. To address this, we carried out a 16-week dietary intervention study where mice were supplemented with a MFGM/EV-rich concentrate, a control diet supplemented with a whey protein concentrate and devoid of milk lipids, or regular chow. Assessment of commonly used markers of metabolic health, including body weight, glucose intolerance and liver microanatomy, demonstrated no differences across the dietary regimes. In contrast, in-depth lipidomic analysis revealed accretion of milk-derived very long odd-chain sphingomyelins and ceramides in blood plasma and multiple tissues of mice fed the MFGM/EV diet. Furthermore, lipidomic flux analysis uncovered that mice fed the MFGM/EV diet have increased lipid metabolic turnover at the whole-body level. These findings help fill a long-lasting knowledge gap between the intake of MFGM/EV-containing foods and the health-promoting effects of their lipid constituents. In addition, the findings suggest that dietary sphingomyelins or ceramide-breakdown products with very long-chains can be used as structural components of cellular membranes, lipoprotein particles and signaling molecules that modulate metabolic homeostasis and health.

FastDFE

Sendrowski, J., Bataillon, T. — Wed, 01 May 2024 05:08:20 +0200

Estimating the distribution of fitness effects (DFE) of new mutations is of fundamental importance in evolutionary biology, ecology, and conservation. However, existing methods for DFE estimation suffer from limitations, such as slow computation speed and limited scalability. To address these issues, we introduce fastDFE, a Python-based software package, offering fast, and flexible DFE inference from site-frequency spectrum (SFS) data. Apart from providing efficient joint inference of multiple DFEs that share parameters, it offers the feature of introducing genomic covariates that influence the DFEs and testing their significance. To further simplify usage, fastDFE is equipped with comprehensive VCF-To-SFS parsing utilities. These include options for site filtering and stratification, as well as site-degeneracy annotation and probabilistic ancestral-Allele inference. fastDFE thereby covers the entire workflow of DFE inference from the moment of acquiring a raw VCF file. Despite its Python foundation, fastDFE incorporates a full R interface, including native R visualization capabilities. The package is comprehensively tested and documented at fastdfe.readthedocs.io.

CryoEM reconstructions of membrane proteins solved in several amphipathic solvents, nanodisc, amphipol and detergents, yield amphipathic belts of similar sizes corresponding to a common ordered solvent layer

Zampieri, V., Gobet, A., Robert, X., Falson, P., Chaptal, V. — Mon, 01 Nov 2021 05:08:20 +0100

To maintain membrane proteins soluble in aqueous solution, amphipathic compounds are used to shield the hydrophobic patch of their membrane insertion, which forms a belt around the protein. This amphipathic belt is seldom looked at due to the difficulty to visualize it. Cryo-EM is now offering this possibility, where belts are visible in 3D reconstructions. We investigated membrane proteins solved in nanodiscs, amphipols or detergents to analyze whether the nature of the amphipathic compound influences the belt size in 3D reconstructions. We identified belt boundaries in map-density distributions and measured distances for every reconstruction. We showed that all the belts create on average similar reconstructions, whether they originate from the same protein, or from protein from different shapes and structures. There is no difference among detergents or types of nanodisc used. These observations illustrate that the belt observed in 3D reconstructions corresponds to the minimum ordered layer around membrane proteins.

Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter

Chaptal, V., Zampieri, V., Wiseman, B., et al. — Fri, 28 Jan 2022 05:08:20 +0100

Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg2+-bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis. Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.

Overexpression of the ABC Transporter BmrA Within Intracellular Caveolae in Escherichia coli

Gobet, A., Jaxel, C., Magnard, S., et al. — Sat, 01 Jan 2022 05:08:20 +0100

We describe here the overproduction and oriented membrane insertion of membrane protein inside intracellular vesicles named heterologous caveolae within E. coli. The method is described with BmrA, a multidrug efflux pump from Bacillus subtilis. BmrA is produced in these vesicles, thanks to the coexpression with the canine caveolin-1β, one of the two isoforms of caveolin-1. Enriched by sucrose gradient, the caveolae-containing fraction allows to probe the ATPase and Hoechst 33342 transport activities, the latter displaying a higher specific activity than the same without caveolin-1β.

The non-Newtonian behavior of detergents during concentration is increased by macromolecules, in trans, and results in their over-concentration

Wed, 01 Feb 2023 05:08:20 +0100

Concentration of pure membrane proteins in detergent solution results in detergent concentration, albeit in unknown amounts. This phenomenon is observed in every lab working on membrane proteins, but has seldom been investigated. In this study, we explored the behavior of detergents mixed with membrane proteins during the step of sample concentration using centrifugal devices. We show that detergent over-concentrate with the presence of polymers, typically membrane or soluble proteins but also polysaccharides. The over-concentration of detergents depends on centrifugal force applied to the device. With the use of a specific dye, we observed the formation of a mesh on the concentrator device. Importantly, reducing the centrifugal speed allows to reduce the concentration of detergents when mixed to macromolecules, as tested with 3 different membrane proteins. All together, these results highlight the non-Newtonian behavior of detergents and provides a solid framework to investigators to improve drastically biochemical and structural studies of membrane proteins.

Conformational space exploration of cryo-EM structures by variability refinement

Afonine, P. V., Gobet, A., Moissonnier, L., Martin, J., Poon, B. K., Chaptal, V. — Sat, 01 Apr 2023 05:08:20 +0200

Cryo-EM observation of biological samples enables visualization of sample heterogeneity, in the form of discrete states that are separable, or continuous heterogeneity as a result of local protein motion before flash freezing. Variability analysis of this continuous heterogeneity describes the variance between a particle stack and a volume, and results in a map series describing the various steps undertaken by the sample in the particle stack. While this observation is absolutely stunning, it is very hard to pinpoint structural details to elements of the maps. In order to bridge the gap between observation and explanation, we designed a tool that refines an ensemble of structures into all the maps from variability analysis. Using this bundle of structures, it is easy to spot variable parts of the structure, as well as the parts that are not moving. Comparison with molecular dynamics simulations highlights the fact that the movements follow the same directions, albeit with different amplitudes. Ligand can also be investigated using this method. Variability refinement is available in the Phenix software suite, accessible under the program name phenix.varref.

CryoEM Data Analysis of Membrane Proteins. Practical Considerations on Amphipathic Belts, Ligands, and Variability Analysis

Gobet, A., Moissonnier, L., Chaptal, V. — Mon, 01 Jan 2024 05:08:20 +0100

Membrane proteins data analysis by cryoEM shows some specificities, as can be found in other typical investigations such as biochemistry, biophysics, or X-ray crystallography. Membrane proteins are typically surrounded by an amphipathic belt that will have some degree of influence on the 3D reconstruction and analysis. In this chapter, we review our experience with the ABC transporter BmrA, as well as our statistical analysis of amphipathic belts around membrane proteins, to bring awareness on some particular features of membrane protein investigations by cryoEM.

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains

Di Cesare, M., Kaplan, E., Rendon, J., et al. — Mon, 01 Jan 2024 05:08:20 +0100

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Feed intake in housed dairy cows

Giagnoni, G., Lassen, J., Lund, P., Foldager, L., Johansen, M., Weisbjerg, M. R. — Sat, 01 Jun 2024 05:08:20 +0200

Measuring feed intake accurately is crucial to determine feed efficiency and for genetic selection. A system using three-dimensional (3D) cameras and deep learning algorithms can measure the volume of feed intake in dairy cows, but for now, the system has not been validated for feed intake expressed as weight of feed. The aim of this study was to validate the weight of feed intake predicted from the 3D cameras with the actual measured weight. It was hypothesised that diet−specific coefficients are necessary for predicting changes in weight, that the relationship between weight and volume is curvilinear throughout the day, and that manually pushing the feed affects this relationship. Twenty-four lactating Danish Holstein cows were used in a cross-over design with four dietary treatments, 2 × 2 factorial arranged with either grass-clover silage or maize silage as silage factor, and barley or dried beet pulp as concentrate factor. Cows were adapted to the diets for 11 d, and for 3 d to tie-stall housing before camera measurements. Six cameras were used for recording, each mounted over an individual feeding platform equipped with a weight scale. When building the predictive models, four cameras were used for training, and the remaining two for testing the prediction of the models. The most accurate predictions were found for the average feed intake over a period when using the starting density of the feed pile, which resulted in the lowest errors, 6% when expressed as RMSE and 5% expressed as mean absolute error. A model including curvilinear effects of feed volume and the impact of manual feed pushing was used on a dataset including daily time points. When cross-validating, the inclusion of a curvilinear effect and a feed push effect did not improve the accuracy of the model for neither the feed pile nor the feed removed by the cow between consecutive time points. In conclusion, measuring daily feed intake from this 3D camera system in the present experimental setup could be accomplished with an acceptable error (below 8%), but the system should be improved for individual meal intake measurements if these measures were to be implemented.

Mining and engineering activity in catalytic amyloids

Peña-Díaz, S., Ferreira, P., Ramos, M. J., Otzen, D. E. — Mon, 01 Jan 2024 05:08:20 +0100

This chapter describes how to test different amyloid preparations for catalytic properties. We describe how to express, purify, prepare and test two types of pathological amyloid (tau and α-synuclein) and two functional amyloid proteins, namely CsgA from Escherichia coli and FapC from Pseudomonas. We therefore preface the methods section with an introduction to these two examples of functional amyloid and their remarkable structural and kinetic properties and high physical stability, which renders them very attractive for a range of nanotechnological designs, both for structural, medical and catalytic purposes. The simplicity and high surface exposure of the CsgA amyloid is particularly useful for the introduction of new functional properties and we therefore provide a computational protocol to graft active sites from an enzyme of interest into the amyloid structure. We hope that the methods described will inspire other researchers to explore the remarkable opportunities provided by bacterial functional amyloid in biotechnology.

Gene expression responses to FUS, EWS, and TAF15 reduction and stress granule sequestration analyses identifies FET-protein non-redundant functions

Blechingberg, J., Luo, Y., Bolund, L., Damgaard, C. K., Nielsen, A. L. — Sun, 01 Jan 2012 05:08:20 +0100

The FET family of proteins is composed of FUS/TLS, EWS/EWSR1, and TAF15 and possesses RNA- and DNA-binding capacities. The FET-proteins are involved in transcriptional regulation and RNA processing, and FET-gene deregulation is associated with development of cancer and protein granule formations in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and trinucleotide repeat expansion diseases. We here describe a comparative characterization of FET-protein localization and gene regulatory functions. We show that FUS and TAF15 locate to cellular stress granules to a larger extend than EWS. FET-proteins have no major importance for stress granule formation and cellular stress responses, indicating that FET-protein stress granule association most likely is a downstream response to cellular stress. Gene expression analyses showed that the cellular response towards FUS and TAF15 reduction is relatively similar whereas EWS reduction resulted in a more unique response. The presented data support that FUS and TAF15 are more functionally related to each other, and that the FET-proteins have distinct functions in cellular signaling pathways which could have implications for the neurological disease pathogenesis.

A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

Fri, 01 Feb 2008 05:08:21 +0100

Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or beta-globin mRNAs, harboring wild-type or various 5' splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5' splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced promoter docking of transcription initiation factors TFIID, TFIIB, and TFIIH on a gene containing a functional 5' splice site. In addition to their promoter association, the TFIID and TFIIH components, TBP and p89, are specifically recruited to the 5' splice site region. Our data suggest a model in which a promoter-proximal 5' splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing pre-initiation complex assembly.

A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model

Krissanaprasit, A., Mihalko, E., Meinhold, K., et al. — Wed, 03 Jul 2024 05:08:21 +0200

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.

Neuroectoderm phenotypes in a human stem cell model of O-GlcNAc transferase associated with intellectual disability

Murray, M., Davidson, L., Ferenbach, A. T., Lefeber, D., van Aalten, D. M.F. — Sat, 01 Jun 2024 05:08:21 +0200

Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation.

Automatic removal of soft tissue from 3D dental photo scans; an important step in automating future forensic odontology identification

Wed, 01 May 2024 05:08:21 +0200

Potentialet af intraorale 3D-fotoscanninger i retsodontologisk identifikation er stort set uudforsket, selvom den høje detaljegrad kunne muliggøre automatisk sammenligning af ante mortem og post mortem tandsæt. Forskelle i blødtvævsforholdene mellem ante- og post mortem intraorale 3D-fotoscanninger kan forårsage tvetydige variationer, hvilket belaster den potentielle automatisering af sammenligningsprocessen og understreger behovet for at begrænse inddragelsen af blødt væv i tandlægesammenligningen. Fjernelsen af blødt væv skal kunne håndtere tandbuer med manglende tænder og intraorale 3D-fotoscanninger, der ikke stammer fra gipsmodeller. For at imødegå disse udfordringer har vi udviklet gitter-skæringsmetoden. Metoden er tilpasselig, hvilket muliggør detaljeret analyse ved hjælp af en lille gitterstørrelse og tilpasning af, hvor meget af det bløde væv der udelukkes fra den beskårne tandscanning. Ved test på 66 tandscanninger var gitter-skæringsmetoden i stand til at begrænse mængden af blødt væv uden at fjerne nogen tænder i 63/66 tandscanninger. De resterende 3 tandscanninger havde delvist frembrudte tredje molarer (visdomstænder), som blev fjernet af gitter-skæringsmetoden. Samlet set repræsenterer gitter-skæringsmetoden et vigtigt skridt mod automatisering af sammenligningsprocessen i retsodontologisk identifikation ved hjælp af intraorale 3D-fotoscanninger.

Population genomics of the muskox' resilience in the near absence of genetic variation

Pečnerová, P., Lord, E., Garcia-Erill, G., et al. — Mon, 01 Jan 2024 05:08:21 +0100

Genomic studies of species threatened by extinction are providing crucial information about evolutionary mechanisms and genetic consequences of population declines and bottlenecks. However, to understand how species avoid the extinction vortex, insights can be drawn by studying species that thrive despite past declines. Here, we studied the population genomics of the muskox (Ovibos moschatus), an Ice Age relict that was at the brink of extinction for thousands of years at the end of the Pleistocene yet appears to be thriving today. We analysed 108 whole genomes, including present-day individuals representing the current native range of both muskox subspecies, the white-faced and the barren-ground muskox (O. moschatus wardi and O. moschatus moschatus) and a ~21,000-year-old ancient individual from Siberia. We found that the muskox' demographic history was profoundly shaped by past climate changes and post-glacial re-colonizations. In particular, the white-faced muskox has the lowest genome-wide heterozygosity recorded in an ungulate. Yet, there is no evidence of inbreeding depression in native muskox populations. We hypothesize that this can be explained by the effect of long-term gradual population declines that allowed for purging of strongly deleterious mutations. This study provides insights into how species with a history of population bottlenecks, small population sizes and low genetic diversity survive against all odds.

A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein

Civit, L., Moradzadeh, N., Jonczyk, A., et al. — Wed, 01 May 2024 05:08:21 +0200

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.