Whether screening the metabolic activity of immune cells facilitates discovery of molecular patho... more Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1–Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.
Using the Swiss Canopy Crane CO 2 enrichment facility, the beech weevil's host choice in a CO 2-e... more Using the Swiss Canopy Crane CO 2 enrichment facility, the beech weevil's host choice in a CO 2-enriched atmosphere and the in situ effect of its infestation on the chemistry of host leaves were investigated. The fraction of infested leaves was larger on CO 2-enriched trees, suggesting that weevil impact on beech may increase as atmospheric CO 2 concentration keeps rising. Egg insertion in the central leaf vein partially interrupts phloem and xylem transport. Because of this separation between the source of photosynthates, the leaf tip, and the rest of the plant, the leaf tip becomes enriched in non-structural carbohydrates and depleted in nitrogen. Elevated CO 2 slightly enhanced non-structural carbohydrates in uninfested leaves, but exerted no further increase in infested leaf part, suggesting a saturation effect. Zusammenfassung Mit Hilfe der, Swiss Canopy Crane' CO 2-Anreicherungsanlage haben wir in situ die Präferenzen der Buchenspringrüssler bei der Wahl ihrer Wirtsbäume nach CO 2-Exposition und den Einfluss ihres Befalls auf die Chemie von Buchenblättern untersucht. An CO 2-behandelten Bäumen war der Befall höher als an Kontrollbäumen. Mit ansteigender atmosphärischer CO 2-Konzentration könnte sich daher die Schadwirkung des Buchenspringrüsslers auf die Rotbuche verstärken. Die Eiablage in der Hauptblattader hat zur Folge, dass Phloem-und Xylemtransport teilweise unterbrochen werden. Diese Unterbrechung zwischen einer Assimilatquelle, der Blattspitze, und dem Rest der Pflanze, führt zu einer Anreicherung nicht-strukturgebundener Kohlenhydrate (NSC) und einer Abnahme der Stickstoff Konzentration in der Blattspitze. Die NSC-Konzentration in nicht befallenen, CO 2-behandelten Blättern zeigte zwar eine leichte Zunahme, stieg aber in den befallenen, CO 2 behandelte Blattspitzen nicht weiter an, was auf einen Sättigungseffekt hindeutet.
The relation between the sequence of a protein and its three-dimensional structure remains largel... more The relation between the sequence of a protein and its three-dimensional structure remains largely unknown. A lasting dream is to elucidate the side-chain-dependent driving forces that govern the folding process. Different structural data suggest that aromatic amino acids play a particular role in the stabilization of protein structures. To better understand the underlying mechanism , we studied peptides of the sequence EGAAXAASS (X = Gly, Ile, Tyr, Trp) through comparison of molecular dynamics (MD) trajectories and NMR residual dipolar coupling (RDC) measurements. The RDC data for aromatic substitutions provide evidence for a kink in the peptide backbone. Analysis of the MD simulations shows that the formation of internal hydrogen bonds underlying a helical turn is key to reproduce the experimental RDC values. The simulations further reveal that the driving force leading to such helical-turn conformations arises from the lack of hydration of the peptide chain on either side of the bulky aromatic side chain, which can potentially act as a nucleation point initiating the folding process. T he prediction of the structural properties of a protein from its amino acid sequence remains a major challenge. 1−3 The detailed mechanism driving the protein folding process is unknown, and specifically its dependence on amino acid side chains. 4 The functional importance of intrinsically disordered proteins has stimulated investigation of the relation between their sequence and their conformational tendency. 5,6 In order to improve predictions about the structure of proteins (folded or disordered), a better understanding of the mechanisms by which individual amino acid side chains impact the conforma-tional dynamics of the protein is required. 4,7 NMR spectroscopy is particularly appropriate to investigate the structural dynamics of peptides in disordered and folded states. 8 Particularly, residual dipolar couplings (RDCs), which arise when molecules are dissolved in anisotropic liquid phases, 9 provide local as well as long-range quantitative structural information on individual chemical bonds. 10−12 The RDC between two nuclei is proportional to the ensemble and time average ⟨(3 cos 2 θ − 1)/2⟩, where θ is the instantaneous angle between the internuclear vector and the magnetic field. In order to investigate the role of individual amino acids on the conformational propensities of a peptide, Dames et al. 13 engineered a series of 14 peptides of sequence EGAAXAASS. The hydrophilic ends ensured solubility, while the nonpolar adjacent residues provided a neutral environment for the systematically single-mutated residue X. They recorded 1 D CαHα and 1 D NH RDCs of these peptides, performing the alignment measurement with polyacrylamide gels. 14 Most peptides (with X = G, I, V, L, N, Q, T, D, E, or K) produced a relatively flat pattern consistent with a rather extended average conformation with little specific local structure. However, the substitutions with the aromatic amino acids Tyr and Trp resulted in a strong reduction of the RDCs or even changes in their signs at the center of the peptide (black lines in Figure S1 in the Supporting Information (SI)), suggesting the presence of a kink at this position. All-atom molecular dynamics (MD) simulations provide the most detailed description of peptide structural dynamics with high spatial and temporal resolution. The NMR data can be used to validate the MD simulation trajectories, which can potentially reveal the mechanistic specificity of aromatic amino acids. Here we show through a systematic comparison between simulated and experimental RDCs that the conformations that best reproduce the experimental data correspond to dynamical ensembles of short helices or turns stabilized by backbone hydrogen bonds. We find that one key driving force that increases the folding propensity of peptides containing aromatic residues arises from the lack of hydration of the carbonyl and amide groups on either side of the bulky hydrophobic side chain. We performed MD simulations in explicit solvent to reproduce previously measured residual dipolar couplings and chemical shifts of peptides of sequence EGAAXAASS. 13 In order to produce adequate sampling, we carried out 7−12 replicated simulations per investigated peptide, each lasting 100 ns. We calculated the 1 D CαHα and 1 D NH RDCs as well as the 1 HN chemical shifts from the coordinates (see Methods in the SI). Figure S1 compares the 1 D CαHα and 1 D NH RDCs, averaged over all of the replicated simulations, and the experimental values published previously. The predicted RDC patterns of the peptide with X = Gly or Ile are relatively flat, accurately reproducing the experimental values, whereas the profiles obtained for X = Tyr or Trp only partially show the RDC variations along the peptide sequence that are observed experimentally. Both the 1 D CαHα and 1 D NH RDCs of the peptides with X = Tyr and Trp fluctuate a lot between the replicated simulations as well as within a given simulation, reflecting the fact that the peptides adopt an ensemble of conformations.
Whether rising atmospheric carbon dioxide (CO2) concentrations will cause forests to grow faster ... more Whether rising atmospheric carbon dioxide (CO2) concentrations will cause forests to grow faster and store more carbon is an open question. Using free air CO2 release in combination with a canopy crane, we found an immediate and sustained enhancement of carbon flux through 35-meter-tall temperate forest trees when exposed to elevated CO2. However, there was no overall stimulation in stem growth and leaf litter production after 4 years. Photosynthetic capacity was not reduced, leaf chemistry changes were minor, and tree species differed in their responses. Although growing vigorously, these trees did not accrete more biomass carbon in stems in response to elevated CO2, thus challenging projections of growth responses derived from tests with smaller trees.
We describe a patient with a heterozygous CTLA4 mutation, associated with low CTLA-4 expression a... more We describe a patient with a heterozygous CTLA4 mutation, associated with low CTLA-4 expression and function of Treg cells, clinically associated with adrenal insufficiency, pure red cell aplasia, and severe T-cell–mediated enterocolitis. The latter was successfully treated with vedolizumab, without apparent side effects. The clinical usefulness of vedolizumab should be assessed further in enterocolitis associated with genetic or drug-induced functional CTLA-4 deficiency.
Flexible polypeptides such as unfolded proteins may access an astronomical number of conformation... more Flexible polypeptides such as unfolded proteins may access an astronomical number of conformations. The most advanced simulations of such states usually comprise tens of thousands of individual structures. In principle, a comparison of parameters predicted from such ensembles to experimental data provides a measure of their quality. In practice, analyses that go beyond the comparison of unbiased average data have been impossible to carry out on the entirety of such very large ensembles and have, therefore, been restricted to much smaller subensembles and/or nondeterministic algorithms. Here, we show that such very large ensembles, on the order of 10 4 to 10 5 conformations, can be analyzed in full by a maximum entropy fit to experimental average data. Maximizing the entropy of the population weights of individual conformations under experimental χ 2 constraints is a convex optimization problem, which can be solved in a very efficient and robust manner to a unique global solution even for very large ensembles. Since the population weights can be determined reliably, the reweighted full ensemble presents the best model of the combined information from simulation and experiment. Furthermore, since the reduction of entropy due to the experimental constraints is well-defined, its value provides a robust measure of the information content of the experimental data relative to the simulated ensemble and an indication for the density of the sampling of conformational space. The method is applied to the reweighting of a 35 000 frame molecular dynamics trajectory of the nonapeptide EGAAWAASS by extensive NMR 3 J coupling and RDC data. The analysis shows that RDCs provide significantly more information than 3 J couplings and that a discontinuity in the RDC pattern at the central tryptophan is caused by a cluster of helical conformations. Reweighting factors are moderate and consistent with errors in MD force fields of less than 3kT. The required reweighting is larger for an ensemble derived from a statistical coil model, consistent with its coarser nature. We call the method COPER, for convex optimization for ensemble reweighting. Similar advantages of large-scale efficiency and robustness can be obtained for other ensemble analysis methods with convex targets and constraints, such as constrained χ 2 minimization and the maximum occurrence method.
Whether screening the metabolic activity of immune cells facilitates discovery of molecular patho... more Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1–Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.
Using the Swiss Canopy Crane CO 2 enrichment facility, the beech weevil's host choice in a CO 2-e... more Using the Swiss Canopy Crane CO 2 enrichment facility, the beech weevil's host choice in a CO 2-enriched atmosphere and the in situ effect of its infestation on the chemistry of host leaves were investigated. The fraction of infested leaves was larger on CO 2-enriched trees, suggesting that weevil impact on beech may increase as atmospheric CO 2 concentration keeps rising. Egg insertion in the central leaf vein partially interrupts phloem and xylem transport. Because of this separation between the source of photosynthates, the leaf tip, and the rest of the plant, the leaf tip becomes enriched in non-structural carbohydrates and depleted in nitrogen. Elevated CO 2 slightly enhanced non-structural carbohydrates in uninfested leaves, but exerted no further increase in infested leaf part, suggesting a saturation effect. Zusammenfassung Mit Hilfe der, Swiss Canopy Crane' CO 2-Anreicherungsanlage haben wir in situ die Präferenzen der Buchenspringrüssler bei der Wahl ihrer Wirtsbäume nach CO 2-Exposition und den Einfluss ihres Befalls auf die Chemie von Buchenblättern untersucht. An CO 2-behandelten Bäumen war der Befall höher als an Kontrollbäumen. Mit ansteigender atmosphärischer CO 2-Konzentration könnte sich daher die Schadwirkung des Buchenspringrüsslers auf die Rotbuche verstärken. Die Eiablage in der Hauptblattader hat zur Folge, dass Phloem-und Xylemtransport teilweise unterbrochen werden. Diese Unterbrechung zwischen einer Assimilatquelle, der Blattspitze, und dem Rest der Pflanze, führt zu einer Anreicherung nicht-strukturgebundener Kohlenhydrate (NSC) und einer Abnahme der Stickstoff Konzentration in der Blattspitze. Die NSC-Konzentration in nicht befallenen, CO 2-behandelten Blättern zeigte zwar eine leichte Zunahme, stieg aber in den befallenen, CO 2 behandelte Blattspitzen nicht weiter an, was auf einen Sättigungseffekt hindeutet.
The relation between the sequence of a protein and its three-dimensional structure remains largel... more The relation between the sequence of a protein and its three-dimensional structure remains largely unknown. A lasting dream is to elucidate the side-chain-dependent driving forces that govern the folding process. Different structural data suggest that aromatic amino acids play a particular role in the stabilization of protein structures. To better understand the underlying mechanism , we studied peptides of the sequence EGAAXAASS (X = Gly, Ile, Tyr, Trp) through comparison of molecular dynamics (MD) trajectories and NMR residual dipolar coupling (RDC) measurements. The RDC data for aromatic substitutions provide evidence for a kink in the peptide backbone. Analysis of the MD simulations shows that the formation of internal hydrogen bonds underlying a helical turn is key to reproduce the experimental RDC values. The simulations further reveal that the driving force leading to such helical-turn conformations arises from the lack of hydration of the peptide chain on either side of the bulky aromatic side chain, which can potentially act as a nucleation point initiating the folding process. T he prediction of the structural properties of a protein from its amino acid sequence remains a major challenge. 1−3 The detailed mechanism driving the protein folding process is unknown, and specifically its dependence on amino acid side chains. 4 The functional importance of intrinsically disordered proteins has stimulated investigation of the relation between their sequence and their conformational tendency. 5,6 In order to improve predictions about the structure of proteins (folded or disordered), a better understanding of the mechanisms by which individual amino acid side chains impact the conforma-tional dynamics of the protein is required. 4,7 NMR spectroscopy is particularly appropriate to investigate the structural dynamics of peptides in disordered and folded states. 8 Particularly, residual dipolar couplings (RDCs), which arise when molecules are dissolved in anisotropic liquid phases, 9 provide local as well as long-range quantitative structural information on individual chemical bonds. 10−12 The RDC between two nuclei is proportional to the ensemble and time average ⟨(3 cos 2 θ − 1)/2⟩, where θ is the instantaneous angle between the internuclear vector and the magnetic field. In order to investigate the role of individual amino acids on the conformational propensities of a peptide, Dames et al. 13 engineered a series of 14 peptides of sequence EGAAXAASS. The hydrophilic ends ensured solubility, while the nonpolar adjacent residues provided a neutral environment for the systematically single-mutated residue X. They recorded 1 D CαHα and 1 D NH RDCs of these peptides, performing the alignment measurement with polyacrylamide gels. 14 Most peptides (with X = G, I, V, L, N, Q, T, D, E, or K) produced a relatively flat pattern consistent with a rather extended average conformation with little specific local structure. However, the substitutions with the aromatic amino acids Tyr and Trp resulted in a strong reduction of the RDCs or even changes in their signs at the center of the peptide (black lines in Figure S1 in the Supporting Information (SI)), suggesting the presence of a kink at this position. All-atom molecular dynamics (MD) simulations provide the most detailed description of peptide structural dynamics with high spatial and temporal resolution. The NMR data can be used to validate the MD simulation trajectories, which can potentially reveal the mechanistic specificity of aromatic amino acids. Here we show through a systematic comparison between simulated and experimental RDCs that the conformations that best reproduce the experimental data correspond to dynamical ensembles of short helices or turns stabilized by backbone hydrogen bonds. We find that one key driving force that increases the folding propensity of peptides containing aromatic residues arises from the lack of hydration of the carbonyl and amide groups on either side of the bulky hydrophobic side chain. We performed MD simulations in explicit solvent to reproduce previously measured residual dipolar couplings and chemical shifts of peptides of sequence EGAAXAASS. 13 In order to produce adequate sampling, we carried out 7−12 replicated simulations per investigated peptide, each lasting 100 ns. We calculated the 1 D CαHα and 1 D NH RDCs as well as the 1 HN chemical shifts from the coordinates (see Methods in the SI). Figure S1 compares the 1 D CαHα and 1 D NH RDCs, averaged over all of the replicated simulations, and the experimental values published previously. The predicted RDC patterns of the peptide with X = Gly or Ile are relatively flat, accurately reproducing the experimental values, whereas the profiles obtained for X = Tyr or Trp only partially show the RDC variations along the peptide sequence that are observed experimentally. Both the 1 D CαHα and 1 D NH RDCs of the peptides with X = Tyr and Trp fluctuate a lot between the replicated simulations as well as within a given simulation, reflecting the fact that the peptides adopt an ensemble of conformations.
Whether rising atmospheric carbon dioxide (CO2) concentrations will cause forests to grow faster ... more Whether rising atmospheric carbon dioxide (CO2) concentrations will cause forests to grow faster and store more carbon is an open question. Using free air CO2 release in combination with a canopy crane, we found an immediate and sustained enhancement of carbon flux through 35-meter-tall temperate forest trees when exposed to elevated CO2. However, there was no overall stimulation in stem growth and leaf litter production after 4 years. Photosynthetic capacity was not reduced, leaf chemistry changes were minor, and tree species differed in their responses. Although growing vigorously, these trees did not accrete more biomass carbon in stems in response to elevated CO2, thus challenging projections of growth responses derived from tests with smaller trees.
We describe a patient with a heterozygous CTLA4 mutation, associated with low CTLA-4 expression a... more We describe a patient with a heterozygous CTLA4 mutation, associated with low CTLA-4 expression and function of Treg cells, clinically associated with adrenal insufficiency, pure red cell aplasia, and severe T-cell–mediated enterocolitis. The latter was successfully treated with vedolizumab, without apparent side effects. The clinical usefulness of vedolizumab should be assessed further in enterocolitis associated with genetic or drug-induced functional CTLA-4 deficiency.
Flexible polypeptides such as unfolded proteins may access an astronomical number of conformation... more Flexible polypeptides such as unfolded proteins may access an astronomical number of conformations. The most advanced simulations of such states usually comprise tens of thousands of individual structures. In principle, a comparison of parameters predicted from such ensembles to experimental data provides a measure of their quality. In practice, analyses that go beyond the comparison of unbiased average data have been impossible to carry out on the entirety of such very large ensembles and have, therefore, been restricted to much smaller subensembles and/or nondeterministic algorithms. Here, we show that such very large ensembles, on the order of 10 4 to 10 5 conformations, can be analyzed in full by a maximum entropy fit to experimental average data. Maximizing the entropy of the population weights of individual conformations under experimental χ 2 constraints is a convex optimization problem, which can be solved in a very efficient and robust manner to a unique global solution even for very large ensembles. Since the population weights can be determined reliably, the reweighted full ensemble presents the best model of the combined information from simulation and experiment. Furthermore, since the reduction of entropy due to the experimental constraints is well-defined, its value provides a robust measure of the information content of the experimental data relative to the simulated ensemble and an indication for the density of the sampling of conformational space. The method is applied to the reweighting of a 35 000 frame molecular dynamics trajectory of the nonapeptide EGAAWAASS by extensive NMR 3 J coupling and RDC data. The analysis shows that RDCs provide significantly more information than 3 J couplings and that a discontinuity in the RDC pattern at the central tryptophan is caused by a cluster of helical conformations. Reweighting factors are moderate and consistent with errors in MD force fields of less than 3kT. The required reweighting is larger for an ensemble derived from a statistical coil model, consistent with its coarser nature. We call the method COPER, for convex optimization for ensemble reweighting. Similar advantages of large-scale efficiency and robustness can be obtained for other ensemble analysis methods with convex targets and constraints, such as constrained χ 2 minimization and the maximum occurrence method.
Uploads
Papers by Olivier Bignucolo
we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as
a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption
rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome
sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients
with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1–Nrf2 system
to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine
interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified
pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.
we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as
a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption
rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome
sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients
with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1–Nrf2 system
to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine
interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified
pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.