Postnatal persistence of hippocampal Cajal-Retzius cells has a crucial role in the establishment of the hippocampal circuit.

Cajal-Retzius (CR) cells are a transient neuron type that populate the postnatal hippocampus. To understand how the persistence of CR cells influences the maturation of hippocampal circuits, we combined a specific transgenic mouse line with viral vector injection to selectively ablate CR cells from the postnatal hippocampus. We observed layer-specific changes in the dendritic complexity and spine density of CA1 pyramidal cells. In addition, transcriptomic analysis highlighted significant changes in the expression of synapse-related genes across development. Finally, we were able to identify significant changes in the expression levels of latrophilin 2, a postsynaptic guidance molecule known for its role in the entorhinal-hippocampal connectivity. These findings were supported by changes in the synaptic proteomic content in CA1 stratum lacunosum-moleculare. Our results reveal a crucial role for CR cells in the establishment of the hippocampal network.

Burden of disease in myasthenia gravis: taking the patient's perspective.

BACKGROUND: Myasthenia gravis (MG) leads to exertion-dependent muscle weakness, but also psychological and social well-being are limited. We aim to describe the burden of disease in MG including sociodemographic, economical, psychosocial as well as clinical aspects, to compare health-related quality of life (HRQoL) of patients with MG to the general population (genP) and to explore risk factors for a lower HRQoL. METHODS: This case-control study was conducted with MG patients of the German Myasthenia Association. A questionnaire-based survey included sociodemographic and clinical data as well as standardized questionnaires, e.g. the Short Form Health (SF-36). HRQoL was compared to genP in a matched-pairs analysis. Participants of the German Health Interview and Examination Survey for Adults (DEGS1) served as control group. RESULTS: In our study, 1660 MG patients participated and were compared to 2556 controls from the genP. Patients with MG showed lower levels of physical functioning (SF-36 mean 56.0, SD 30.3) compared to the genP (mean 81.8, SD 22.1, adjusted difference: 25, 95% CI 22-29) and lower mental health sub-score (SF-36 mean 67.3, SD 19.8, vs. 74.1, SD 16.7, adjusted difference: 5, 95% CI 2-8). Female gender, higher age, low income, partnership status, lower activities of daily life, symptoms of depression, anxiety and fatigue and self-perceived low social support were associated with a lower HRQoL in MG patients. DISCUSSION: HRQoL is lower in patients with MG compared to genP. The burden of MG on patients includes economic and social aspects as well as their emotional well-being. New therapies must achieve improvements for patients in these areas. TRIAL REGISTRATION INFORMATION: Clinicaltrials.gov, NCT03979521, submitted: June 7, 2019, first patient enrolled: May 1, 2019, https://clinicaltrials.gov/ct2/show/NCT03979521.

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit.

AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.

The enzyme subunit SubA of Shiga toxin-producing E. coli strains demonstrates comparable intracellular transport and cytotoxic activity as the holotoxin SubAB in HeLa and HCT116 cells in vitro.

The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubAΔC344, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.

Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro.

The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 µmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.

Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns.

Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coliIMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.

Toxins of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli.

Studies on Shiga toxin-producing Escherichia coli (STEC) typically examine and classify the virulence gene profiles based on genomic analyses. Among the screened strains, a subgroup of STEC which lacks the locus of enterocyte effacement (LEE) has frequently been identified. This raises the question about the level of pathogenicity of such strains. This review focuses on the advantages and disadvantages of the standard screening procedures in virulence profiling and summarizes the current knowledge concerning the function and regulation of toxins encoded by LEE-negative STEC. Although LEE-negative STEC usually come across as food isolates, which rarely cause infections in humans, some serotypes have been implicated in human diseases. In particular, the LEE-negative E. coli O104:H7 German outbreak strain from 2011 and the Australian O113:H21 strain isolated from a HUS patient attracted attention. Moreover, the LEE-negative STEC O113:H21 strain TS18/08 that was isolated from minced meat is remarkable in that it not only encodes multiple toxins, but in fact expresses three different toxins simultaneously. Their characterization contributes to understanding the virulence of the LEE-negative STEC.

The Chaperone Activity of the Developmental Small Heat Shock Protein Sip1 Is Regulated by pH-Dependent Conformational Changes.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.

Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

Modulation of the Hsp90 chaperone cycle by a stringent client protein.

Hsp90 is the most abundant molecular chaperone in the eukaryotic cell. One of the most stringent clients is the glucocorticoid receptor (GR), whose in vivo function strictly depends on the interaction with the Hsp90 machinery. However, the molecular mechanism of this interaction has been elusive. Here we have reconstituted the interaction of Hsp90 with hormone-bound GR using purified components. Our biochemical and structural analyses define the binding site for GR on Hsp90 and reveal that binding of GR modulates the conformational cycle of Hsp90. FRET experiments demonstrate that a partially closed form of the Hsp90 dimer is the preferred conformation for interaction. Consistent with this, the conformational cycle of Hsp90 is decelerated, and its ATPase activity decreases. Hsp90 cochaperones differentially affect formation of the Hsp90-GR complex, serving as control elements for cycle progression and revealing an intricate interplay of client and cochaperones as molecular modulators of the Hsp90 machine.

Tetramers are the activation-competent species of the HOCl-specific transcription factor HypT.

Hypochlorous acid (HOCl) is an important component of the immune system and is produced by neutrophils to kill invading microorganisms. The transcription factor HypT is specifically activated by HOCl by methionine oxidation and protects Escherichia coli cells from the detrimental effects of HOCl. HypT forms dodecameric ring-like oligomers. Binding of HypT to DNA induces dissociation of the dodecamers into dimers and tetramers, thus forming the DNA-binding species. To dissect HypT dissociation, binding to DNA, and activation, we aimed to dissociate the dodecamers independently of DNA and to analyze HOCl-dependent activation in vitro. We found that HypT dodecamers dissociated into tetramers in the presence of l-arginine and NaCl, which was reversible upon dilution of the additive. Making use of the reversible dissociation, we generated mixed assemblies consisting of wild-type and mutant HypT subunits and determined that mutant subunits with reduced thermal stability were stabilized by wild-type HypT in the mixed assembly. HypT tetramers, as present at high NaCl concentrations, were stabilized against thermal unfolding and aggregation triggered by high HOCl concentrations. Importantly, in vitro activation by HOCl of HypT tetramers was completed within 1 min, whereas activation of dodecamers required 1 h for completion. Furthermore, activation of HypT tetramers required stoichiometric amounts of HOCl instead of an excess of HOCl, as observed for dodecamers. This supports the idea that small HypT oligomers are the activation-competent species, whereas the dodecamers are a storage form. Our study reveals the importance of the dynamic oligomeric state for HypT activation by HOCl.

Role of cysteines in the stability and DNA-binding activity of the hypochlorite-specific transcription factor HypT.

Reactive oxygen species are important components of the immune response. Hypochlorite (HOCl) is produced by neutrophils to kill invading microorganisms. The bactericidal activity of HOCl is due to proteome-wide unfolding and oxidation of proteins at cysteine and methionine residues. Escherichia coli cells are protected from HOCl-killing by the previously identified dodecameric transcription factor HypT (YjiE). Here, we aimed to unravel whether HOCl activates HypT directly or via a reaction product of HOCl with a cellular component. Bacterial viability assays and analysis of target gene regulation indicate that HypT is highly specific to activation by HOCl and that no reaction products of HOCl such as monochloramine, hydroxyl radicals, or methionine sulfoxide activate HypT in vivo. Surprisingly, purified HypT lost its DNA-binding activity upon incubation with HOCl or reaction products that oxidize HypT to form a disulfide-linked dimer, and regained DNA-binding activity upon reduction. Thus, we postulate that the cysteines in HypT contribute to control the DNA-binding activity of HypT in vitro. HypT contains five cysteine residues; a HypT mutant with all cysteines substituted by serine is aggregation-prone and forms tetramers in addition to the typical dodecamers. Using single and multiple cysteine-to-serine mutants, we identified Cys150 to be required for stability and Cys4 being important for oligomerization of HypT to dodecamers. Further, oxidation of Cys4 is responsible for the loss of DNA-binding of HypT upon oxidation in vitro. It appears that Cys4 oxidation upon conditions that are insufficient to stimulate the DNA-binding activity of HypT prevents unproductive interactions of HypT with DNA. Thus, Cys4 oxidation may be a check point in the activation process of HypT.

The small heat shock protein Hsp27 affects assembly dynamics and structure of keratin intermediate filament networks.

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation.

A photoactivatable marker protein for pulse-chase imaging with superresolution.

IrisFP is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green- to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. Here we introduce a monomeric variant, mIrisFP, and demonstrate how its multiple photoactivation modes can be used for pulse-chase experiments combined with subdiffraction-resolution imaging in living cells by using dual-color photoactivation localization microscopy (PALM).