Nico Kümmerer - Academia.edu (original) (raw)

Papers by Nico Kümmerer

Research paper thumbnail of Schematic diagram of the three circuits analysed in this work

<b>Copyright information:</b>Taken from "Noise in transcription negative feedbac... more <b>Copyright information:</b>Taken from "Noise in transcription negative feedback loops: simulation and experimental analysis"Molecular Systems Biology 2006;2():41-41.Published online 01 Aug 2006PMCID:PMC1681513.Copyright © 2006, EMBO and Nature Publishing Group () Negative feedback loop where the TetR protein is fused to GFP (TG-nf) on a low copy plasmid (∼4 copies). () Negative feedback loop, where TetR represses itself and also production of GFP (T+G-nf). TetR is located in a low-copy plasmid (∼4 copies) and the reporter on a medium-copy plasmid (∼60 copies). () TetR is constitutively produced and represses GFP production. TetR is located in a low-copy plasmid (∼4 copies) and the reporter on a medium-copy plasmid (∼60 copies). The circles represent the protein produced: T for TetR, G for GFP and P for polymerase. Promoters are shown as squares: A for the TetR promoter and its regulatory region, C for the GFP promoter and its regulatory region and Z for all other promoters in that could compete for the polymerase. RNA is shown as a wavy line. Arrows mean activation and line ended lines inhibition.

Research paper thumbnail of Experimental and theoretical changes in fluorescence with increasing amounts of aTc

<b>Copyright information:</b>Taken from "Noise in transcription negative feedbac... more <b>Copyright information:</b>Taken from "Noise in transcription negative feedback loops: simulation and experimental analysis"Molecular Systems Biology 2006;2():41-41.Published online 01 Aug 2006PMCID:PMC1681513.Copyright © 2006, EMBO and Nature Publishing Group () Mean fluorescence values measured by FACS for the TG-nf circuit (○), the T+G-nf circuit (◊) and T+G circuit (□). () Number of GFP molecules calculated with the simulations for the TG-nf circuit (○), the T+G-nf circuit (◊) and T+G circuit (□). In both cases, values for the TG-nf circuit are plotted on a secondary -axis for clarity.

Research paper thumbnail of Article number: 41

www.molecularsystemsbiology.com

Research paper thumbnail of FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system

Proceedings of the National Academy of Sciences, 2010

Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. F... more Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 Å resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the bi...

Research paper thumbnail of Structural basis for the molecular evolution of SRP-GTPase activation by protein

Nature Structural & Molecular Biology, 2011

Hybrid assay. Upper panel: The proteins p53 and SV40IT were used as controls. Full-length FlhF in... more Hybrid assay. Upper panel: The proteins p53 and SV40IT were used as controls. Full-length FlhF interacts with YlxH, however only in one direction, which suggests a sterical hindrance by the N-terminal B-domain. This effect is not observed for the interaction of NG-FlhF with YlxH. Lower panel: Fulllength FlhF shows a weak self-interaction. Combination of FlhF and NG-FlhF shows significant stronger interactions. NG-FlhF shows a strong selfinteraction. These observations suggest a regulatory role of the B-domain on FlhF homodimer formation as suggested previously 1 and are reminiscent to the situation of the A-domain in the SRP receptor FtsY 2. No self-interaction of YlxH was observed.

Research paper thumbnail of Noise in transcription negative feedback loops: simulation and experimental analysis

Molecular Systems Biology, 2006

Negative feedback loops have been invoked as a way to control and decrease transcriptional noise.... more Negative feedback loops have been invoked as a way to control and decrease transcriptional noise. Here, we have built three circuits to test the effect of negative feedback loops on transcriptional noise of an autoregulated gene encoding a transcription factor (TF) and a downstream gene (DG), regulated by this TF. Experimental analysis shows that self-repression decreases noise compared to expression from a non-regulated promoter. Interestingly enough, we find that noise minimization by negative feedback loop is optimal within a range of repression strength. Repression values outside this range result in noise increase producing a U-shaped behaviour. This behaviour is the result of external noise probably arising from plasmid fluctuations as shown by simulation of the network. Regarding the target gene of a self-repressed TF (sTF), we find a strong decrease of noise when repression by the sTF is strong and a higher degree of noise anti-correlation between sTF and its target. Simulations of the circuits indicate that the main source of noise in these circuits could come from plasmid variation and therefore that negative feedback loops play an important role in suppressing both external and internal noise. An important observation is that DG expression without negative feedback exhibits bimodality at intermediate TF repression values. This bimodal behaviour seems to be the result of external noise as it can only be found in those simulations that include plasmid variation.

Research paper thumbnail of Amyloid Toxicity Is Independent of Polypeptide Sequence, Length and Chirality

Journal of Molecular Biology, 2008

By using an amyloid sequence pattern, here we have identified putative sixresidue amyloidogenic s... more By using an amyloid sequence pattern, here we have identified putative sixresidue amyloidogenic stretches in several relevant amyloid proteins. Hexapeptides synthesized on the bases of the sequence stretches matching the pattern have been shown to form amyloid fibrils in vitro. As larger pathological peptides such as Aβ 1-42 do, these short amyloid peptides form heterogeneous mixtures of small aggregates that induce cell death in PC12 cells and primary hippocampal neurons. Toxic mixtures of small aggregates from these hexapeptides bind to cell membranes and can be further internalized, as also observed for natural amyloid proteins. In neurons, toxic aggregates obtained from the full length Aβ 1-42 amyloid peptide or their amyloid stretch Aβ 16-21 peptide preferentially localize in synapses, leading to the reorganization of the underlying actin cytoskeleton. This process does not involve stereospecific interactions between membrane and toxic species as D-sequences are as toxic as L ones, suggesting that is not receptor mediated. Based on these results, we propose here that regardless of polypeptide sequence, length and amino acid chirality, amyloid prefibrillar aggregates exert their cytotoxic effect through a common cell death mechanism related to a particular quaternary structure. The degree of toxicity of these species seems to depend, however, on cell membrane composition.

Research paper thumbnail of Schematic diagram of the three circuits analysed in this work

<b>Copyright information:</b>Taken from "Noise in transcription negative feedbac... more <b>Copyright information:</b>Taken from "Noise in transcription negative feedback loops: simulation and experimental analysis"Molecular Systems Biology 2006;2():41-41.Published online 01 Aug 2006PMCID:PMC1681513.Copyright © 2006, EMBO and Nature Publishing Group () Negative feedback loop where the TetR protein is fused to GFP (TG-nf) on a low copy plasmid (∼4 copies). () Negative feedback loop, where TetR represses itself and also production of GFP (T+G-nf). TetR is located in a low-copy plasmid (∼4 copies) and the reporter on a medium-copy plasmid (∼60 copies). () TetR is constitutively produced and represses GFP production. TetR is located in a low-copy plasmid (∼4 copies) and the reporter on a medium-copy plasmid (∼60 copies). The circles represent the protein produced: T for TetR, G for GFP and P for polymerase. Promoters are shown as squares: A for the TetR promoter and its regulatory region, C for the GFP promoter and its regulatory region and Z for all other promoters in that could compete for the polymerase. RNA is shown as a wavy line. Arrows mean activation and line ended lines inhibition.

Research paper thumbnail of Experimental and theoretical changes in fluorescence with increasing amounts of aTc

<b>Copyright information:</b>Taken from "Noise in transcription negative feedbac... more <b>Copyright information:</b>Taken from "Noise in transcription negative feedback loops: simulation and experimental analysis"Molecular Systems Biology 2006;2():41-41.Published online 01 Aug 2006PMCID:PMC1681513.Copyright © 2006, EMBO and Nature Publishing Group () Mean fluorescence values measured by FACS for the TG-nf circuit (○), the T+G-nf circuit (◊) and T+G circuit (□). () Number of GFP molecules calculated with the simulations for the TG-nf circuit (○), the T+G-nf circuit (◊) and T+G circuit (□). In both cases, values for the TG-nf circuit are plotted on a secondary -axis for clarity.

Research paper thumbnail of Article number: 41

www.molecularsystemsbiology.com

Research paper thumbnail of FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system

Proceedings of the National Academy of Sciences, 2010

Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. F... more Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 Å resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the bi...

Research paper thumbnail of Structural basis for the molecular evolution of SRP-GTPase activation by protein

Nature Structural & Molecular Biology, 2011

Hybrid assay. Upper panel: The proteins p53 and SV40IT were used as controls. Full-length FlhF in... more Hybrid assay. Upper panel: The proteins p53 and SV40IT were used as controls. Full-length FlhF interacts with YlxH, however only in one direction, which suggests a sterical hindrance by the N-terminal B-domain. This effect is not observed for the interaction of NG-FlhF with YlxH. Lower panel: Fulllength FlhF shows a weak self-interaction. Combination of FlhF and NG-FlhF shows significant stronger interactions. NG-FlhF shows a strong selfinteraction. These observations suggest a regulatory role of the B-domain on FlhF homodimer formation as suggested previously 1 and are reminiscent to the situation of the A-domain in the SRP receptor FtsY 2. No self-interaction of YlxH was observed.

Research paper thumbnail of Noise in transcription negative feedback loops: simulation and experimental analysis

Molecular Systems Biology, 2006

Negative feedback loops have been invoked as a way to control and decrease transcriptional noise.... more Negative feedback loops have been invoked as a way to control and decrease transcriptional noise. Here, we have built three circuits to test the effect of negative feedback loops on transcriptional noise of an autoregulated gene encoding a transcription factor (TF) and a downstream gene (DG), regulated by this TF. Experimental analysis shows that self-repression decreases noise compared to expression from a non-regulated promoter. Interestingly enough, we find that noise minimization by negative feedback loop is optimal within a range of repression strength. Repression values outside this range result in noise increase producing a U-shaped behaviour. This behaviour is the result of external noise probably arising from plasmid fluctuations as shown by simulation of the network. Regarding the target gene of a self-repressed TF (sTF), we find a strong decrease of noise when repression by the sTF is strong and a higher degree of noise anti-correlation between sTF and its target. Simulations of the circuits indicate that the main source of noise in these circuits could come from plasmid variation and therefore that negative feedback loops play an important role in suppressing both external and internal noise. An important observation is that DG expression without negative feedback exhibits bimodality at intermediate TF repression values. This bimodal behaviour seems to be the result of external noise as it can only be found in those simulations that include plasmid variation.

Research paper thumbnail of Amyloid Toxicity Is Independent of Polypeptide Sequence, Length and Chirality

Journal of Molecular Biology, 2008

By using an amyloid sequence pattern, here we have identified putative sixresidue amyloidogenic s... more By using an amyloid sequence pattern, here we have identified putative sixresidue amyloidogenic stretches in several relevant amyloid proteins. Hexapeptides synthesized on the bases of the sequence stretches matching the pattern have been shown to form amyloid fibrils in vitro. As larger pathological peptides such as Aβ 1-42 do, these short amyloid peptides form heterogeneous mixtures of small aggregates that induce cell death in PC12 cells and primary hippocampal neurons. Toxic mixtures of small aggregates from these hexapeptides bind to cell membranes and can be further internalized, as also observed for natural amyloid proteins. In neurons, toxic aggregates obtained from the full length Aβ 1-42 amyloid peptide or their amyloid stretch Aβ 16-21 peptide preferentially localize in synapses, leading to the reorganization of the underlying actin cytoskeleton. This process does not involve stereospecific interactions between membrane and toxic species as D-sequences are as toxic as L ones, suggesting that is not receptor mediated. Based on these results, we propose here that regardless of polypeptide sequence, length and amino acid chirality, amyloid prefibrillar aggregates exert their cytotoxic effect through a common cell death mechanism related to a particular quaternary structure. The degree of toxicity of these species seems to depend, however, on cell membrane composition.