Thioredoxin is a metabolic rheostat controlling regulatory B cells.

Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (Breg) cell differentiation and function is unknown. Here we show that Breg cell differentiation, unlike non-Breg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by Breg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing Breg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by Breg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx+ B cells. Exogenous Trx stimulation restored Breg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies Breg cell impairment in patients with SLE.

Inactive disease in patients with lupus is linked to autoantibodies to type I interferons that normalize blood IFNα and B cell subsets.

Systemic lupus erythematosus (SLE) is characterized by increased expression of type I interferon (IFN)-regulated genes in 50%-75% of patients. We report that out of 501 patients with SLE analyzed, 73 (14%) present autoantibodies against IFNα (anti-IFN-Abs). The presence of neutralizing-anti-IFN-Abs in 4.2% of patients inversely correlates with low circulating IFNα protein levels, inhibition of IFN-I downstream gene signatures, and inactive global disease score. Hallmarks of SLE pathogenesis, including increased immature, double-negative plasmablast B cell populations and reduction in regulatory B cell (Breg) frequencies, were normalized in patients with neutralizing anti-IFN-Abs compared with other patient groups. Immunoglobulin G (IgG) purified from sera of patients with SLE with neutralizing anti-IFN-Abs impedes CpGC-driven IFNα-dependent differentiation of B cells into immature B cells and plasmablasts, thus recapitulating the neutralizing effect of anti-IFN-Abs on B cell differentiation in vitro. Our findings highlight a role for neutralizing anti-IFN-Abs in controlling SLE pathogenesis and support the use of IFN-targeting therapies in patients with SLE lacking neutralizing-anti-IFN-Abs.

Role of CD14+ monocyte-derived oxidised mitochondrial DNA in the inflammatory interferon type 1 signature in juvenile dermatomyositis.

OBJECTIVES: To define the host mechanisms contributing to the pathological interferon (IFN) type 1 signature in Juvenile dermatomyositis (JDM). METHODS: RNA-sequencing was performed on CD4+, CD8+, CD14+ and CD19+ cells sorted from pretreatment and on-treatment JDM (pretreatment n=10, on-treatment n=11) and age/sex-matched child healthy-control (CHC n=4) peripheral blood mononuclear cell (PBMC). Mitochondrial morphology and superoxide were assessed by fluorescence microscopy, cellular metabolism by 13C glucose uptake assays, and oxidised mitochondrial DNA (oxmtDNA) content by dot-blot. Healthy-control PBMC and JDM pretreatment PBMC were cultured with IFN-α, oxmtDNA, cGAS-inhibitor, TLR-9 antagonist and/or n-acetyl cysteine (NAC). IFN-stimulated gene (ISGs) expression was measured by qPCR. Total numbers of patient and controls for functional experiments, JDM n=82, total CHC n=35. RESULTS: Dysregulated mitochondrial-associated gene expression correlated with increased ISG expression in JDM CD14+ monocytes. Altered mitochondrial-associated gene expression was paralleled by altered mitochondrial biology, including 'megamitochondria', cellular metabolism and a decrease in gene expression of superoxide dismutase (SOD)1. This was associated with enhanced production of oxidised mitochondrial (oxmt)DNA. OxmtDNA induced ISG expression in healthy PBMC, which was blocked by targeting oxidative stress and intracellular nucleic acid sensing pathways. Complementary experiments showed that, under in vitro experimental conditions, targeting these pathways via the antioxidant drug NAC, TLR9 antagonist and to a lesser extent cGAS-inhibitor, suppressed ISG expression in pretreatment JDM PBMC. CONCLUSIONS: These results describe a novel pathway where altered mitochondrial biology in JDM CD14+ monocytes lead to oxmtDNA production and stimulates ISG expression. Targeting this pathway has therapeutical potential in JDM and other IFN type 1-driven autoimmune diseases.

Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes.

Genome duplication is safeguarded by constantly adjusting the activity of the replicative CMG (CDC45-MCM2-7-GINS) helicase. However, minichromosome maintenance proteins (MCMs)-the structural core of the CMG helicase-have never been visualized at sites of DNA synthesis inside a cell (the so-called MCM paradox). Here, we solve this conundrum by showing that anti-MCM antibodies primarily detect inactive MCMs. Upon conversion of inactive MCMs to CMGs, factors that are required for replisome activity bind to the MCM scaffold and block MCM antibody binding sites. Tagging of endogenous MCMs by CRISPR-Cas9 bypasses this steric hindrance and enables MCM visualization at active replisomes. Thus, by defining conditions for detecting the structural core of the replicative CMG helicase, our results explain the MCM paradox, provide visual proof that MCMs are an integral part of active replisomes in vivo, and enable the investigation of replication dynamics in living cells exposed to a constantly changing environment.

ReconSil: An electron microscopy toolbox to study helicase function at an origin of replication.

The loading of the MCM replicative helicase onto eukaryotic origins of replication occurs via a sequential, symmetric mechanism. Here, we describe a method to study this multistep reaction using electron microscopy. Tools presented include protein expression and purification protocols, methods to produce asymmetric replication origin substrates and bespoke image processing strategies. DNA templates include recognisable protein roadblocks that help to orient DNA replication factors along a specific origin sequence. Detailed electron microscopy image processing protocols are provided to reposition 2D averages onto the original micrograph for the in silico reconstitution of fully occupied origins of replication. Using these tools, a chemically trapped helicase loading intermediate is observed sliding along origin DNA, showcasing a key feature of the MCM loading mechanism. Although developed to study replicative helicase loading, this method can be employed to investigate the mechanism of other multicomponent biochemical reactions, occurring on a flexible polymeric substrate.

Differences in the metabolic and functional mechanisms used to tolerate flooding in Guazuma ulmifolia (Lam.) from flood-prone Amazonian and dry Cerrado savanna populations.

Flood tolerance is crucial to the survival of tree species subject to long periods of flooding, such as those present in the Amazonian várzea. Tolerance can be mediated by adjustments of metabolism, physiology and morphology, reinforcing the need to investigate the physiological and biochemical mechanisms used by tropical tree species to survive this stress. Moreover, such mechanisms may vary between populations that are subjected to differences in the frequency of flooding events. Here, we aimed to identify the mechanisms used by two populations of the tropical tree Guazuma ulmifolia (Lam.) to tolerate flooding: an Amazonian population frequently exposed to flooding and a Cerrado population, adapted to a dry environment. Young plants were subjected to a flooding of the roots and lower stem for 32 days, followed by 17 days of recovery. Amazonian plants exhibited greater increases in shoot length and higher maximum photosynthetic rate (Amax) compared with non-flooded plants from 7 days of flooding onwards, whereas increased Amax occurred later in flooded Cerrado plants and was not accompanied by increased shoot length. Lactate accumulated in roots of Cerrado plants after 24 h flooding, together with transcripts coding for lactate dehydrogenase in roots of both Cerrado and Amazonian plants. After 7 days of flooding, lactate decreased and alcohol dehydrogenase activity increased transiently, together with concentrations of alanine, γ-aminobutyric acid and succinate, indicating activation of metabolic processes associated with low oxygen availability. Other amino acids also increased in flooded Cerrado plants, revealing more extensive metabolic changes than in Amazonian plants. Wetland and dryland populations of G. ulmifolia revealed the great capacity to tolerate flooding stress through a suite of alterations in photosynthetic gas exchange and metabolism. However, the integrated physiological, biochemical and molecular analyses realized here indicated that wetland plants acclimatized more efficiently with increased shoot elongation and more rapid restoration of normal metabolism.

Structural and Biophysical Insights into SPINK1 Bound to Human Cationic Trypsin.

(1) The serine protease inhibitor Kazal type 1 (SPINK1) inhibits trypsin activity in zymogen granules of pancreatic acinar cells. Several mutations in the SPINK1 gene are associated with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The most common variant is SPINK1 p.N34S. Although this mutation was identified two decades ago, the mechanism of action has remained elusive. (2) SPINK1 and human cationic trypsin (TRY1) were expressed in E. coli, and inhibitory activities were determined. Crystals of SPINK1-TRY1 complexes were grown by using the hanging-drop method, and phases were solved by molecular replacement. (3) Both SPINK1 variants show similar inhibitory behavior toward TRY1. The crystal structures are almost identical, with minor differences in the mutated loop. Both complexes show an unexpected rotamer conformation of the His63 residue in TRY1, which is a member of the catalytic triad. (4) The SPINK1 p.N34S mutation does not affect the inhibitory behavior or the overall structure of the protein. Therefore, the pathophysiological mechanism of action of the p.N34S variant cannot be explained mechanistically or structurally at the protein level. The observed histidine conformation is part of a mechanism for SPINK1 that can explain the exceptional proteolytic stability of this inhibitor.

PEGylated Domain I of Beta-2-Glycoprotein I Inhibits Thrombosis in a Chronic Mouse Model of the Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disorder in which autoantibodies cause clinical effects of vascular thrombosis and pregnancy morbidity. The only evidence-based treatments are anticoagulant medications such as warfarin and heparin. These medications have a number of disadvantages, notably risk of haemorrhage. Therefore, there is a pressing need to develop new, more focused treatments that target the actual pathogenic disease process in APS. The pathogenic antibodies exert their effects by interacting with phospholipid-binding proteins, of which the most important is beta-2-glycoprotein I. This protein has five domains, of which the N-terminal Domain I (DI) is the main site for binding of pathogenic autoantibodies. We previously demonstrated bacterial expression of human DI and showed that this product could inhibit the ability of IgG from patients with APS (APS-IgG) to promote thrombosis in a mouse model. Since DI is a small 7kDa protein, its serum half-life would be too short to be therapeutically useful. We therefore used site-specific chemical addition of polyethylene glycol (PEG) to produce a larger variant of DI (PEG-DI) and showed that PEG-DI was equally effective as the non-PEGylated DI in inhibiting thrombosis caused by passive transfer of APS-IgG in mice. In this paper, we have used a mouse model that reflects human APS much more closely than the passive transfer of APS-IgG. In this model, the mice are immunized with human beta-2-glycoprotein I and develop endogenous anti-beta-2-glycoprotein I antibodies. When submitted to a pinch stimulus at the femoral vein, these mice develop clots. Our results show that PEG-DI inhibits production of thromboses in this model and also reduces expression of tissue factor in the aortas of the mice. No toxicity was seen in mice that received PEG-DI. Therefore, these results provide further evidence supporting possible efficacy of PEG-DI as a potential treatment for APS.

In silico reconstitution of DNA replication. Lessons from single-molecule imaging and cryo-tomography applied to single-particle cryo-EM.

DNA replication has been reconstituted in vitro with yeast proteins, and the minimal system requires the coordinated assembly of 16 distinct replication factors, consisting of 42 polypeptides. To understand the molecular interplay between these factors at the single residue level, new structural biology tools are being developed. Inspired by advances in single-molecule fluorescence imaging and cryo-tomography, novel single-particle cryo-EM experiments have been used to characterise the structural mechanism for the loading of the replicative helicase. Here, we discuss how in silico reconstitution of single-particle cryo-EM data can help describe dynamic systems that are difficult to approach with conventional three-dimensional classification tools.

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

Current Challenges in Plant Systems Biology.

Plants, as biological systems, are organized and regulated by a complex network of interactions from the genetic to the morphological level and suffer substantial influence from the environment. Reductionist approaches have been widely used in plant biology but have failed to reveal the mechanisms by which plants can growth under adverse conditions. It seems likely, therefore, that to understand the complexity of plant metabolic responses it is necessary to adopt non-reductionist approaches such as those from systems biology. Although such approaches seem methodologically complex to perform and difficult to interpret, they have been successfully applied in both metabolic and gene expression networks in a wide range of microorganisms and more recently in plants. Given the advance of techniques that allow complex analysis of plant cells, high quantities of data are currently generated and are available for in silico analysis and mathematical modeling. It is increasingly recognized, therefore, that the use of different methods such as graph analysis and dynamic network modeling are needed to better understand this abundance of information. However, before these practical advances, one of the main challenges currently in plant biology is to change the paradigm from the classical reductionism to the systemic level, which requires not only scientific but also educational changes.

Characterization of raffinose metabolism genes uncovers a wild Arachis galactinol synthase conferring tolerance to abiotic stresses.

Raffinose family oligosaccharides (RFOs) are implicated in plant regulatory mechanisms of abiotic stresses tolerance and, despite their antinutritional proprieties in grain legumes, little information is available about the enzymes involved in RFO metabolism in Fabaceae species. In the present study, the systematic survey of legume proteins belonging to five key enzymes involved in the metabolism of RFOs (galactinol synthase, raffinose synthase, stachyose synthase, alpha-galactosidase, and beta-fructofuranosidase) identified 28 coding-genes in Arachis duranensis and 31 in A. ipaënsis. Their phylogenetic relationships, gene structures, protein domains, and chromosome distribution patterns were also determined. Based on the expression profiling of these genes under water deficit treatments, a galactinol synthase candidate gene (AdGolS3) was identified in A. duranensis. Transgenic Arabidopsis plants overexpressing AdGolS3 exhibited increased levels of raffinose and reduced stress symptoms under drought, osmotic, and salt stresses. Metabolite and expression profiling suggested that AdGolS3 overexpression was associated with fewer metabolic perturbations under drought stress, together with better protection against oxidative damage. Overall, this study enabled the identification of a promising GolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in crops, whilst also contributing to the understanding of RFO metabolism in legume species.

Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM.

In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA1,2. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer3. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites4,5. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX.

Ubiquitin C-terminal hydrolase deubiquitinase BAP1 is an essential tumor suppressor involved in cell growth control, DNA damage response, and transcriptional regulation. As part of the Polycomb repression machinery, BAP1 is activated by the deubiquitinase adaptor domain of ASXL1 mediating gene repression by cleaving ubiquitin (Ub) from histone H2A in nucleosomes. The molecular mechanism of BAP1 activation by ASXL1 remains elusive, as no structures are available for either BAP1 or ASXL1. Here, we present the crystal structure of the BAP1 ortholog from Drosophila melanogaster, named Calypso, bound to its activator, ASX, homolog of ASXL1. Based on comparative structural and functional analysis, we propose a model for Ub binding by Calypso/ASX, uncover decisive structural elements responsible for ASX-mediated Calypso activation, and characterize the interaction with ubiquitinated nucleosomes. Our results give molecular insight into Calypso function and its regulation by ASX and provide the opportunity for the rational design of mechanism-based therapeutics to treat human BAP1/ASXL1-related tumors.

PEGylated Domain I of Beta-2-Glycoprotein I Inhibits the Binding, Coagulopathic, and Thrombogenic Properties of IgG From Patients With the Antiphospholipid Syndrome.

APS is an autoimmune disease in which antiphospholipid antibodies (aPL) cause vascular thrombosis and pregnancy morbidity. In patients with APS, aPL exert pathogenic actions by binding serum beta-2-glycoprotein I (ß2GPI) via its N-terminal domain I (DI). We previously showed that bacterially-expressed recombinant DI inhibits biological actions of IgG derived from serum of patients with APS (APS-IgG). DI is too small (7 kDa) to be a viable therapeutic agent. Addition of polyethylene glycol (PEGylation) to small molecules enhances the serum half-life, reduces proteolytic targeting and can decrease immunogenicity. It is a common method of tailoring pharmacokinetic parameters and has been used in the production of many therapies in the clinic. However, PEGylation of molecules may reduce their biological activity, and the size of the PEG group can alter the balance between activity and half-life extension. Here we achieve production of site-specific PEGylation of recombinant DI (PEG-DI) and describe the activities in vitro and in vivo of three variants with different size PEG groups. All variants were able to inhibit APS-IgG from: binding to whole ß2GPI in ELISA, altering the clotting properties of human plasma and promoting thrombosis and tissue factor expression in mice. These findings provide an important step on the path to developing DI into a first-in-class therapeutic in APS.

Label-free quantitative proteomics of rat hypothalamus under fever induced by LPS and PGE2.

Fever is a brain-mediated increase in body temperature mainly during inflammatory or infectious challenges. Although there is considerable data regarding the inflammation pathways involved in fever, metabolic alterations necessary to orchestrate the complex inflammatory response are not totally understood. We performed proteomic analysis of rat hypothalamus using label-free LC-MS/MS in a model of fever induced by lipopolysaccharide (LPS) or prostaglandin E2 (PGE2). In total, 7021 proteins were identified. As far as we know, this is the largest rat hypothalamus proteome dataset available to date. Pathway analysis showed proteins from both stimuli associated with inflammatory and metabolic pathways. Concerning metabolic pathways, rats exposed to LPS or PGE2 presented lower relative abundance of proteins involved in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle. Mitochondrial function may also be altered by both stimuli because significant downregulation of several proteins was found, mainly in complexes I and IV. LPS was able to induce downregulation of important proteins in the enzymatic antioxidant system, thereby contributing to oxidative stress. The results offered comprehensive information about fever responses and helped to reveal new insights into proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during systemic LPS and central PGE2 administration. SIGNIFICANCE: The evolutionary persistence of fever, despite the elevated cost for maintenance of this response, suggests that elevation in core temperature may represent an interesting strategy for survival. Fever response is achieved through the integrated behavioral, physiological, immunological and biochemical processes that determine the balance between heat generation and elimination. The development of such complex response arouses interest in studying how the cell metabolism responds or even contributes to promote fever. Our results offered comprehensive information about fever responses, including metabolic and inflammatory pathways, providing new insights into candidate proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during fever induced by systemic LPS and central PGE2 perturbation.

Gas Chromatography-Mass Spectrometry-Based 13C-Labeling Studies in Plant Metabolomics.

Stable-isotope labeling analysis has been used to discover new metabolic pathways and their key regulatory points in a wide range of organisms. Given the complexity of the plant metabolic network, this analysis provides information complementary to that obtained from metabolite profiling that can be used to understand how plants cope with adverse conditions, and how metabolism varies between different cells, tissues, and organs. Here we describe the experimental procedures from sample harvesting and extraction to mass spectral analysis and interpretation that allow the researcher to perform 13C-labeling experiments. A wide range of plant material, from single cells to whole plants, can be used to investigate the metabolic fate of the 13C from a predefined tracer. Thus, a key point of this analysis is to choose the correct biological system, the substrate and the condition to be investigated; all of which implicitly relies on the biological question to be investigated. Rapid sample quenching and a careful data analysis are also critical points in such studies. By contrast to other metabolomic approaches, stable-isotope labeling can provide information concerning the fluxes through metabolic networks, which is essential for understanding and manipulating metabolic phenotypes and therefore of pivotal importance for both systems biology and plant metabolic engineering.

Going viral in rheumatology: using social media to show that mechanistic research is relevant to patients with lupus and antiphospholipid syndrome.

OBJECTIVES: There is a lack of published data regarding patient interaction in basic scientific research, including methodologies for simple, cost-effective interactions and the outcomes of such studies. Therefore, we aimed to evaluate the ease of generating patient opinion data on specific scientific research projects whilst establishing a template for other groups to follow. Our secondary objective was to assess which research topics are of most interest to patients with SLE and/or APS. METHODS: Through patient-based interactions, we developed a lay summary of a mechanistic research proposal and a set of associated questions to assess patient opinion on this research topic. We disseminated the questions as an online survey with associated lay summary through patient-based charity websites and social media. The survey was open for 3 weeks. RESULTS: Of 527 respondents, 520 reported having SLE or APS. The patient response to the research proposal was overwhelmingly positive, with the majority expressing strong interest in the mechanistic aspect of the project. Analysis of free text box responses confirmed that the most popular research topics for patients were as follows: treatment, genetics, triggers, diagnosis and mechanistic research. Interestingly, patient interest in disease mechanisms featured more frequently than clinical topics, such as management of disease flares. CONCLUSION: It is possible to conduct short-term, valuable patient engagement at low cost, using an online survey and social media. This methodology may form a good template for future patient engagement. The volume and distribution of positive response shows that patients are interested in mechanistic research.