Cytokine levels in the gingival crevicular fluid and their association with periodontal status of down syndrome patients: a cross-sectional study.

OBJECTIVE: To evaluate cytokine levels of interleukin (IL)-1ß, IL-4, IL-6, IL-17a, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the gingival crevicular fluid (GCF) of periodontal sites in individuals with Down syndrome (DS) and analyze their relationship with clinical periodontal parameters. MATERIALS AND METHODS: A cross-sectional study was conducted with 49 DS patients and 32 individuals without DS (non-DS group). Periodontal probing depth (PPD), clinical attachment level (CAL), bleeding on probing (BoP), and visible plaque index (VPI) were evaluated. The periodontal sites were classified as shallow, moderate, and deep. GCF was collected in all shallow sites and, when present, in moderate and deep sites for the analysis of cytokine levels. The cytokines, IL-1ß, IL-4, IL-6, IL-17a, TNF-α, and IFN-γ, were quantified using the Luminex® automatic analyzer system. RESULTS: The DS group presented greater severity of periodontitis compared to the non-DS group (P = 0.005). The DS group showed a significant direct correlation of IL-1ß and an inverse correlation of IFN-γ and IL-14 with all periodontal variables. In the analysis stratified by periodontal pocket depth, we observed a higher level of IFN-γ, IL-17a, IL-1ß, and IL-6 in the shallow sites, and IL-17a, IL-1ß, and IL-6 in deep pockets of DS group individuals. Multivariate models showed that higher levels of IL-1ß, IL-4, IL-6, and IL-17a were associated with Down syndrome even after adjusting for periodontal status, sex, and age. CONCLUSION: The findings suggest that people with DS have greater periodontal impairment and higher levels of cytokines in GCF, even in sites having clinical periodontal parameters similar to those of individuals without DS. These data reiterate the concept of an altered and less effective immune response in the population with DS in the face of a periodontal microbial challenge. CLINICAL RELEVANCE: Elevated periodontal inflammation burden can be observed with higher cytokine levels in the gingival crevicular fluid of people with Down syndrome, especially IL-1, IL-4, IL-6, and IL-17, regardless of the stage of periodontitis.

Accurate selected ion flow tube mass spectrometry quantification of ethylene oxide contamination in the presence of acetaldehyde.

In September 2020, traces of ethylene oxide (a toxic substance used as a pesticide in developing countries but banned for use on food items within the European Union) were found in foodstuffs containing ingredients derived from imported sesame seed products. Vast numbers of foodstuffs were recalled across Europe due to this contamination, leading to expensive market losses and extensive trace exposure of ethylene oxide to consumers. Therefore, a rapid analysis method is needed to ensure food safety by high-throughput screening for ethylene oxide contamination. Selected ion flow tube mass spectrometry (SIFT-MS) is a suitable method for rapid quantification of trace amounts of vapours in the headspace of food samples. It turns out, however, that the presence of acetaldehyde complicates SIFT-MS analyses of its isomer ethylene oxide. It was proposed that a combination of the H3O+ and NO+ reagent ions can be used to analyse ethylene oxide in the presence of acetaldehyde. This method is, however, not robust because of the product ion overlaps and potential interferences from other matrix species. Thus, we studied the kinetics of the reactions of the H3O+, NO+, OH- and O-˙ ions with these two compounds and obtained their rate coefficients and product ion branching ratios. Interpretation of these experimental data revealed that the OH- anions are the most suitable SIFT-MS reagents because the product ions of their reactions with acetaldehyde (CH2CHO- at m/z 43) and ethylene oxide (C2H3O2- at m/z 59) do not overlap.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

The influence of periodontal status and serum biomarkers on salivary leptin levels in systemic lupus erythematosus patients.

Objective: This study aimed to investigate the influence of periodontal status, clinical data, and serum markers on salivary leptin levels in patients with systemic lupus erythematosus (SLE). Methods: A case-control study was conducted with 38 patients with SLE and 29 healthy controls. Periodontal data included periodontal probing depth (PPD), clinical attachment level (CAL), and gingival bleeding on probing (BOP). Stimulated saliva samples were collected to analyze salivary leptin levels. Clinical and serum data were collected from the SLE group. Statistical analysis included the t-test, Mann-Whitney test, Spearman correlation coefficient, and a structural equation model. Results: The SLE group had a lower salivary leptin level than the control group (P = 0.002). The model revealed that SLE had an inverse and independent effect on salivary leptin (standardized estimate =  - 0.289, P = 0.023). Moreover, salivary leptin level negatively correlated with the serum levels of triglyceride, creatinine, and leukocytes, positively correlated with the serum total cholesterol, but was not significantly correlated with the periodontal status. Conclusion: These findings suggest that patients with SLE have a lower salivary leptin level. In addition, the level of salivary leptin does not appear to be related to periodontal status in patients with SLE.

Identifying metabolic parameters related to severity and extent of periodontitis in down syndrome patients.

BACKGROUND AND OBJECTIVE: Systemic metabolic status and periodontitis can be related in patients with Down syndrome (DS). The objective of this study was to identify metabolic indicators (anthropometric measurements, blood pressure, and serum markers) related to severity and extent of periodontitis in DS patients. METHODS: A cross-sectional study was conducted with 49 patients with DS. Periodontal evaluation included the periodontal probing depth (PPD), clinical attachment level (CAL), gingival bleeding index (GBI), and visible plaque index (VPI). Periodontitis severity was classified by the stages system. The extent of periodontitis was assessed as the percentage of sites with CAL ≥3 mm, CAL ≥4 mm, PPD ≥4 mm, and PPD ≥5 mm. The metabolic condition of the participants was determined by analyzing anthropometric parameters, blood pressure, and serum markers. Data were analyzed using the Mann-Whitney test, Spearman's correlation coefficient, and multivariate regression analysis. RESULTS: Periodontitis stage 3/4 was detected in 32.7% of patients, with high values of VPI (54.6 ± 35.8) and GBI (42.4 ± 33.3). The severity of periodontitis was related to higher mean corpuscular hemoglobin (ß = .291, p = .028) and mean corpuscular volume values (ß = .293, p = .046). Arm circumference measurements were inversely related to CAL ≥3 mm (ß = -.408, p = .023), PPD ≥4 mm (ß = -.475, p = .006), and PPD ≥5 mm (ß = -.443, p = .010). CONCLUSIONS: The findings suggest that the severity and extent of periodontitis may be related to some metabolic parameters in DS patients.

Protist impacts on marine cyanovirocell metabolism.

The fate of oceanic carbon and nutrients depends on interactions between viruses, prokaryotes, and unicellular eukaryotes (protists) in a highly interconnected planktonic food web. To date, few controlled mechanistic studies of these interactions exist, and where they do, they are largely pairwise, focusing either on viral infection (i.e., virocells) or protist predation. Here we studied population-level responses of Synechococcus cyanobacterial virocells (i.e., cyanovirocells) to the protist Oxyrrhis marina using transcriptomics, endo- and exo-metabolomics, photosynthetic efficiency measurements, and microscopy. Protist presence had no measurable impact on Synechococcus transcripts or endometabolites. The cyanovirocells alone had a smaller intracellular transcriptional and metabolic response than cyanovirocells co-cultured with protists, displaying known patterns of virus-mediated metabolic reprogramming while releasing diverse exometabolites during infection. When protists were added, several exometabolites disappeared, suggesting microbial consumption. In addition, the intracellular cyanovirocell impact was largest, with 4.5- and 10-fold more host transcripts and endometabolites, respectively, responding to protists, especially those involved in resource and energy production. Physiologically, photosynthetic efficiency also increased, and together with the transcriptomics and metabolomics findings suggest that cyanovirocell metabolic demand is highest when protists are present. These data illustrate cyanovirocell responses to protist presence that are not yet considered when linking microbial physiology to global-scale biogeochemical processes.

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan.

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing "Candidatus Thermoflexus japonica," "Candidatus Thermoflexus tengchongensis," and "Candidatus Thermoflexus sinensis." Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

Early changes in IL-21, IL-22, CCL2, and CCL4 serum cytokines after outpatient autologous transplantation for multiple sclerosis: A proof of concept study.

Changes in serum cytokines after autologous hematopoietic stem cell transplantation (AHSCT) in multiple sclerosis (MS) patients were documented. Thirty-six consecutive MS patients who had their Expanded Disability Status Scale (EDSS) scored before AHSCT were prospectively enrolled. Cyclophosphamide (Cy) was infused at 200 mg/kg in two administrations given 10 days apart: the first dose for mobilization, the second as the conditioning regimen. Patients were mobilized with 10 µg/kg/day subcutaneous G-CSF. Serum was collected 14 days before and 14 after AHSCT. IL-6, IL-9, IL-10, IL 17-A, IL-21, IL-22, IL-23, TNF-A, CCL2, CCL3, and CCL4 were measured by magnetic bead-based immunoassay. t Test and Wilcoxon test were used to compare cytokine levels before and after AHSCT. There were 28 women and 8 men with a median age of 46 (15-62) years, median duration of MS was 9.5 (1-32) years, and EDSS score was 5.7 (1.5-8.0). Patients had a decrement of pro-inflammatory IL-21 and IL-22 (p = .003 and p = .028) and an increment of anti-inflammatory CCL2 and CCL4 (p < .001 and p = .039) after AHSCT. Decrease of IL-21 and IL-22 coupled with an increment of CCL2 and CCL4 could reflect the immunomodulatory effect of auto-HSCT and be an early indicator of its efficacy.

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

An integrated workflow for phenazine-modifying enzyme characterization.

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

The prognostic significance of serum XCL1 concentration in patients with acute lymphoblastic leukemia: a pilot study.

There is no information about XCL1 in patients with acute lymphoblastic leukemia (ALL). The objective of this study was to correlate the serum levels of XCL1 and survival in ALL patients. Only ALL patients older than 12 months were considered to participate. Serum XCL1 was measured at diagnosis, end of remission induction, and end of consolidation. Thirty-three ALL patients with median age of 21 years (1-78) were included. Higher XCL1 level (above 50 pg/mL) at ALL diagnosis correlated with higher survival (p = 0.038), whereas XCL1 level at end of induction and consolidation had no significant correlation. Concerning the behavior of serum XCL1 during treatment, higher survival at 5 years was observed in the group with progressively decreased levels of XCL1 (70%) than those with progressively increasing (29%) or no detectable XCL1 (14%). In conclusion, higher serum XCL1 levels at diagnosis and their progressive decline throughout chemotherapy could be correlated with higher survival.

TNF-α increases in the CSF of children with acute lymphoblastic leukemia before CNS relapse.

There is scarce information regarding the concentration of cytokines in cerebrospinal fluid (CSF) of children with acute lymphoblastic leukemia (ALL) and their clinical association with CNS status. A prospective analysis of 40 patients <18years with newly diagnosed ALL was performed. Human cytokine magnetic bead panel assay values of IL-2, IL-4, IL-6, IL-8, IL-10, MCP-1, TNF-α in CSF at diagnosis, end of induction to remission, and 6months after diagnosis were determined. IL-6 and MCP-1 values showed a significant increment at the end of induction. From the whole group 4 (10.0%), patients relapsed to the CNS at a median of 11.48months. A significantly higher value of TNF-α at third determination in these CNS-relapsed patients was documented, 7.48 vs. 2.86pg/mL in 36 children without relapse (p=0.024). TNF-α concentration increased at a median 5.48months before CNS relapse. By receiver-operating characteristic curve (ROC) analysis, the best cut-off point of TNF-α concentration that better predicted CNS relapse was ≥1.79pg/mL. In conclusion an increase in TNF-α concentration on CSF preceded CNS relapse in children with ALL. An increase in MCP-1 and IL-6 was not associated to CNS relapse and appears to result from an inflammatory response after IT injection of chemotherapy.

Exometabolomics and MSI: deconstructing how cells interact to transform their small molecule environment.

Metabolism is at the heart of many biotechnologies from biofuels to medical diagnostics. Metabolomic methods that provide glimpses into cellular metabolism have rapidly developed into a critical component of the biotechnological development process. Most metabolomics methods have focused on what is happening inside the cell. Equally important are the biochemical transformations of the cell, and their effect on other cells and their environment; the exometabolome. Exometabolomics is therefore gaining popularity as a robust approach for obtaining rich phenotypic data, and being used in bioprocessing and biofuel development. Mass spectrometry imaging approaches, including several nanotechnologies, provide complimentary information by localizing metabolic processes within complex biological matrices. Together, the two technologies can provide new insights into the metabolism and interactions of cells.

Benchtop isolation and characterization of functional exosomes by sequential filtration.

Early and minimally invasive detection of malignant events or other pathologies is of utmost importance in the pursuit of improved patient care and outcomes. Recent evidence indicates that exosomes and extracellular vesicles in serum and body fluids can contain nucleic acid, protein, and other biomarkers. Accordingly, there is great interest in applying these clinically as prognostic, predictive, pharmacodynamic, and early detection indicators. Nevertheless, existing exosome isolation methods can be time-consuming, require specialized equipment, and/or present other inefficiencies regarding purity, reproducibility and assay cost. We have developed a straightforward, three-step protocol for exosome isolation of cell culture supernatants or large volumes of biofluid based on sequential steps of dead-end pre-filtration, tangential flow filtration (TFF), and low-pressure track-etched membrane filtration that we introduce here. Our approach yields exosome preparations of high purity and defined size distribution and facilitates depletion of free protein and other low-molecular-weight species, extracellular vesicles larger than 100nm, and cell debris. Samples of exosomes prepared using the approach were verified morphologically by nanoparticle tracking analysis and electron microscopy, and mass spectrometry analyses confirmed the presence of previously reported exosome-associated proteins. In addition to being easy-to-implement, sequential filtration yields exosomes of high purity and, importantly, functional integrity as a result of the relatively low-magnitude manipulation forces employed during isolation. This answers an unmet need for preparation of minimally manipulated exosomes for investigations into exosome function and basic biology. Further, the strategy is amenable to translation for clinical exosome isolations because of its speed, automatability, scalability, and specificity for isolating exosomes from complex biological samples.

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells.

L-asparaginase (L-ASP) is a therapeutic enzyme used clinically for the treatment of childhood acute lymphoblastic leukemia. L-ASP's anticancer activity is believed to be associated primarily with depletion of asparagine, but secondary glutaminase activity has also been implicated in its anticancer mechanism of action. To investigate the effects of L-ASP on amino acid metabolism, we have developed an LC-MS/MS metabolomics platform for high-throughput quantitation of 29 metabolites, including all 20 proteinogenic amino acids, 6 metabolically related amino acid derivatives (ornithine, citrulline, sarcosine, taurine, hypotaurine, and cystine), and 3 polyamines (putrescince, spermidine, and spermine) in adherent cultured cells. When we examined the response of OVCAR-8 ovarian cancer cells in culture to L-ASP, asparagine was depleted from the medium within seconds. Interestingly, intracellular asparagine was also depleted rapidly, and the mechanism was suggested to involve rapid export of intracellular asparagine followed by rapid conversion to aspartic acid by L-ASP. We also found that L-ASP-induced cell death was more closely associated with glutamine concentration than with asparagine concentration. Time-course analysis revealed the dynamics of amino acid metabolism after feeding cells with fresh medium. Overall, this study provides new insight into L-ASP's mechanism of action, and the optimized analytical method can be extended, with only slight modification, to other metabolically active amino acids, related compounds, and a range of cultured cell types.

An artifact in LC-MS/MS measurement of glutamine and glutamic acid: in-source cyclization to pyroglutamic acid.

Advances in metabolomics, particularly for research on cancer, have increased the demand for accurate, highly sensitive methods for measuring glutamine (Gln) and glutamic acid (Glu) in cell cultures and other biological samples. N-terminal Gln and Glu residues in proteins or peptides have been reported to cyclize to pyroglutamic acid (pGlu) during liquid chromatography (LC)-mass spectrometry (MS) analysis, but cyclization of free Gln and Glu to free pGlu during LC-MS analysis has not been well-characterized. Using an LC-MS/MS protocol that we developed to separate Gln, Glu, and pGlu, we found that free Gln and Glu cyclize to pGlu in the electrospray ionization source, revealing a previously uncharacterized artifact in metabolomic studies. Analysis of Gln standards over a concentration range from 0.39 to 200 µM indicated that a minimum of 33% and maximum of almost 100% of Gln was converted to pGlu in the ionization source, with the extent of conversion dependent on fragmentor voltage. We conclude that the sensitivity and accuracy of Gln, Glu, and pGlu quantitation by electrospray ionization-based mass spectrometry can be improved dramatically by using (i) chromatographic conditions that adequately separate the three metabolites, (ii) isotopic internal standards to correct for in-source pGlu formation, and (iii) user-optimized fragmentor voltage for acquisition of the MS spectra. These findings have immediate impact on metabolomics and metabolism research using LC-MS technologies.

Measurement of DNA concentration as a normalization strategy for metabolomic data from adherent cell lines.

Metabolomics is a rapidly advancing field, and much of our understanding of the subject has come from research on cell lines. However, the results and interpretation of such studies depend on appropriate normalization of the data; ineffective or poorly chosen normalization methods can lead to frankly erroneous conclusions. That is a recurrent challenge because robust, reliable methods for normalization of data from cells have not been established. In this study, we have compared several methods for normalization of metabolomic data from cell extracts. Total protein concentration, cell count, and DNA concentration exhibited strong linear correlations with seeded cell number, but DNA concentration was found to be the most generally useful method for the following reasons: (1) DNA concentration showed the greatest consistency across a range of cell numbers; (2) DNA concentration was the closest to proportional with cell number; (3) DNA samples could be collected from the same dish as the metabolites; and (4) cell lines that grew in clumps were difficult to count accurately. We therefore conclude that DNA concentration is a widely applicable method for normalizing metabolomic data from adherent cell lines.

Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation.

Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood. We show that SEX4 switches between reduced (active) and oxidized (inactive) states, suggesting that SEX4 is redox-regulated. Although only partial reactivation of SEX4 was achieved using artificial reductants (e.g. dithiothreitol), use of numerous chloroplastic thioredoxins recovered activity completely, suggesting that thioredoxins could reduce SEX4 in vivo. Analysis of peptides from oxidized and reduced SEX4 identified a disulfide linkage between the catalytic cysteine at position 198 (Cys198) and the cysteine at position 130 (Cys130) within the phosphatase domain. The position of these cysteines was structurally analogous to that for known redox-regulated dual-specificity phosphatases, suggesting a common mechanism of reversible oxidation amongst these phosphatases. Mutation of Cys130 renders SEX4 more sensitive to oxidative inactivation and less responsive to reductive reactivation. Together, these results provide the first biochemical evidence for a redox-dependent structural switch that regulates SEX4 activity, which represents the first plant phosphatase known to undergo reversible oxidation via disulfide bond formation like its mammalian counterparts.

The membrane-binding domain of an amphitropic enzyme suppresses catalysis by contact with an amphipathic helix flanking its active site.

CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in the synthesis of phosphatidylcholine, is activated by binding membranes using a lipid-induced amphipathic helix (domain M). Domain M functions to silence catalysis when CCT is not membrane engaged. The silencing mechanism is unknown. We used photo-cross-linking and mass spectrometry to identify contacts between domain M and other CCT domains in its soluble form. Each of four sites in domain M forged cross-links to the same set of peptides that flank the active site and overlap at helix αE at the base of the active site. These cross-links were broken in the presence of activating lipid vesicles. Mutagenesis of domain M revealed that multiple hydrophobic residues within a putative auto-inhibitory (AI) motif contribute to the contact with helix αE and silencing. Helix αE was confirmed as the docking site for domain M by deuterium exchange analysis. We compared the dynamics and fold stability of CCT domains by site-directed fluorescence anisotropy and urea denaturation. The results suggest a bipartite structure for domain M: a disordered N-terminal portion and an ordered C-terminal AI motif with an unfolding transition identical with that of helix αE. Reduction in hydrophobicity of the AI motif decreased its order and fold stability, as did deletion of the catalytic domain. These results support a model in which catalytic silencing is mediated by the docking of an amphipathic AI motif onto the amphipathic helices αE. An unstructured leash linking αE with the AI motif may facilitate both the silencing contact and its membrane-triggered disruption.

Assembly of Ebola virus matrix protein VP40 is regulated by latch-like properties of N and C terminal tails.

The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers) but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.