Kristoffer Brännström

The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

Structural basis of signal-sequence recognition by the signal recognition particle

The signal recognition particle (SRP) recognizes and binds the signal sequence of nascent proteins as they emerge from the ribosome. We present here the 3.0-Å structure of a signal sequence bound to the Methanococcus jannaschii SRP core. Structural comparison with the free SRP core shows that signal-sequence binding induces formation of the GM-linker helix and a 180° flip of the NG domain—structural changes that ensure a hierarchical succession of events during protein targeting.

Promiscuous cross-seeding between bacterial amyloids promotes interspecies biofilms

Background: Seeding of eukaryotic amyloids is highly specific. Results: Curli subunits from different bacteria can cross-seed and such interspecies interactions restore surface attachment and biofilm formation. Conclusion: Curli cross-seeding is relaxed, which promotes interspecies biofilms. Significance: This is the first study on cross-seeding of bacterial amyloids and will help define the roles of bacterial amyloids in multispecies biofilms. Amyloids are highly aggregated proteinaceous fibers historically associated with neurodegenerative conditions including Alzheimers, Parkinsons, and prion-based encephalopathies. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding. Seeding of disease-associated amyloids and prions is highly specific and cross-seeding is usually limited or prevented. Here we describe the first study on the cross-seeding potential of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development. Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. Our results showed that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2, and Citrobacter koseri were able to cross-seed in vitro. The polymerization of Escherichia coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Cross-seeding of curli proteins was also observed in mixed colony biofilms with E. coli and S. typhimurium. CsgA was secreted from E. coli csgB− mutants assembled into fibers on adjacent S. typhimurium that presented CsgB on its surfaces. Similarly, CsgA was secreted by S. typhimurium csgB− mutants formed curli on CsgB-presenting E. coli. This interspecies curli assembly enhanced bacterial attachment to agar surfaces and supported pellicle biofilm formation. Collectively, this work suggests that the seeding specificity among curli homologs is relaxed and that heterogeneous curli fibers can facilitate multispecies biofilm development.

Amyloid-β Oligomer Specificity Mediated by the IgM Isotype – Implications for a Specific Protective Mechanism Exerted by Endogenous Auto-Antibodies

Background Alzheimers disease (AD) has been strongly linked to an anomalous self-assembly of the amyloid-β peptide (Aβ). The correlation between clinical symptoms of AD and Aβ depositions is, however, weak. Instead small and soluble Aβ oligomers are suggested to exert the major pathological effects. In strong support of this notion, immunological targeting of Aβ oligomers in AD mice-models shows that memory impairments can be restored without affecting the total burden of Aβ deposits. Consequently a specific immunological targeting of Aβ oligomers is of high therapeutic interest. Methodology/Principal Findings Previously the generation of conformational-dependent oligomer specific anti-Aβ antibodies has been described. However, to avoid the difficult task of identifying a molecular architecture only present on oligomers, we have focused on a more general approach based on the hypothesis that all oligomers expose multiple identical epitopes and therefore would have an increased binding to a multivalent receptor. Using the polyvalent IgM immunoglobulin we have developed a monoclonal anti-Aβ antibody (OMAB). OMAB only demonstrates a weak interaction with Aβ monomers and dimers having fast on and off-rate kinetics. However, as an effect of avidity, its interaction with Aβ-oligomers results in a strong complex with an exceptionally slow off-rate. Through this mechanism a selectivity towards Aβ oligomers is acquired and OMAB fully inhibits the cytotoxic effect exerted by Aβ(1-42) at highly substoichiometric ratios. Anti-Aβ auto-antibodies of IgM isotype are frequently present in the sera of humans. Through a screen of endogenous anti-Aβ IgM auto-antibodies from a group of healthy individuals we show that all displays a preference for oligomeric Aβ. Conclusions/Significance Taken together we provide a simple and general mechanism for targeting of oligomers without the requirement of conformational-dependent epitopes. In addition, our results suggest that IgM anti-Aβ auto-antibodies may exert a more specific protective mechanism in vivo than previously anticipated.

Properties of RNA Polymerase Bypass Mutants IMPLICATIONS FOR THE ROLE OF ppGpp AND ITS CO-FACTOR DksA IN CONTROLLING TRANSCRIPTION DEPENDENT ON σ54

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain σ70-dependent promoters. A number of promoters that are dependent on alternative σ-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived σ54-dependent Po promoter and several other σ54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro σ54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the β- and β′-subunits of core RNA polymerase that allow high levels of transcription from the σ54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with σ-factors to favor σ54-holoenzyme formation over that with σ70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of σ54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent σ70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative σ-factors is controlled to meet cellular requirements.

The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling.

ABSTRACT The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

The C-terminal repeating units of CsgB direct bacterial functional amyloid nucleation

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β-strand-loop-β-strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the first, second, or third repeating units were able to convert CsgA into fibers. However, mutants missing either the fourth or fifth repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2, and 4 self-assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.

Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori.

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

The N-terminal Region of Amyloid β Controls the Aggregation Rate and Fibril Stability at Low pH Through a Gain of Function Mechanism

Alzheimers disease is linked to a pathological polymerization of the endogenous amyloid β-peptide (Aβ) that ultimately forms amyloid plaques within the human brain. We used surface plasmon resonance (SPR) to measure the kinetic properties of Aβ fibril formation under different conditions during the polymerization process. For all polymerization processes, a critical concentration of free monomers, as defined by the dissociation equilibrium constant (K(D)), is required for the buildup of the polymer, for example, amyloid fibrils. At concentrations below the K(D), polymerization cannot occur. However, the K(D) for Aβ has previously been shown to be several orders of magnitude higher than the concentrations found in the cerebrospinal and interstitial fluids of the human brain, and the mechanism by which Aβ amyloid forms in vivo has been a matter of debate. Using SPR, we found that the K(D) of Aβ dramatically decreases as a result of lowering the pH. Importantly, this effect enables Aβ to polymerize within a picomolar concentration range that is close to the physiological Aβ concentration within the human brain. The stabilizing effect is dynamic, fully reversible, and notably pronounced within the pH range found within the endosomal and lysosomal pathways. Through sequential truncation, we show that the N-terminal region of Aβ contributes to the enhanced fibrillar stability due to a gain of function mechanism at low pH. Our results present a possible route for amyloid formation at very low Aβ concentrations and raise the question of whether amyloid formation in vivo is restricted to a low pH environment. These results have general implications for the development of therapeutic interventions.

A Generic Method for Design of Oligomer-Specific Antibodies

Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e.g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the Aβ peptide and α-synuclein that are associated with Alzheimers and Parkinsons diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.