Assembly of the Tn7 targeting complex by a regulated stepwise process.

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.

Inhibition of BACE1 affected both its Aß producing and degrading activities and increased Aß42 and Aß40 levels at high level BACE1 expression.

The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the predominant ß-secretase, cleaving the amyloid precursor protein (APP) via the amyloidogenic pathway. In addition, BACE1 as an amyloid degrading enzyme (ADE), cleaves Aß to produce the C-terminally truncated non-toxic Aß fragment Aß34 which is an indicator of amyloid clearance. Here, we analyzed effects of BACE1 inhibitors on its opposing enzymatic functions, i.e., amyloidogenic (Aß producing) and amyloidolytic (Aß degrading) activities, using cell culture models with varying BACE1/APP ratios. Under high level BACE1 expression, low-dose inhibition unexpectedly yielded a two-fold increase in Aß42 and Aß40 levels. The concomitant decrease in Aß34 and secreted APPß levels suggested that the elevated Aß42 and Aß40 levels were due to the attenuated Aß degrading activity of BACE1. Notably, the amyloidolytic activity of BACE1 was impeded at lower BACE1 inhibitor concentrations compared to its amyloidogenic activity, thereby suggesting that the Aß degrading activity of BACE1 was more sensitive to inhibition than its Aß producing activity. Under endogenous BACE1 and APP levels, "low-dose" BACE1 inhibition affected both the Aß producing and degrading activities of BACE1, i.e., significantly increased Aß42/Aß40 ratio and decreased Aß34 levels, respectively. Further, we incubated recombinant BACE1 with synthetic Aß peptides and found that BACE1 has higher affinity for Aß substrates over APP. In summary, our results suggest that stimulating BACE1's ADE activity and halting Aß production without decreasing Aß clearance could still be a promising therapeutic approach with new, yet to be developed, BACE1 modulators.

Identification and characterization of calreticulin as a novel plasminogen receptor.

Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.

The amyloid-ß1-42-oligomer interacting peptide D-AIP possesses favorable biostability, pharmacokinetics, and brain region distribution.

We have previously developed a unique 8-amino acid Aß42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aß42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aß42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aß42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease.

Small-Molecule Ligands that Bind the RET Receptor Activate Neuroprotective Signals Independent of but Modulated by Coreceptor GFRα1.

Glial cell line-derived neurotrophic factor (GDNF) binds the GFRα1 receptor, and the GDNF-GFRα1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFRα1-RET signaling complex, agents that bind and activate RET directly and independently of GFRα1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFRα1 coexpression. However, RET activation by these ligands is constrained by GFRα1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT: A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor α, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.

Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells.

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein-coupled receptor (GPCR) leading to PLCß activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Label-free distribution of anti-amyloid D-AIP in Drosophila melanogaster: prevention of Aß42-induced toxicity without side effects in transgenic flies.

Soluble oligomers of the 42-amino acid amyloid beta (Aß42) peptide are highly toxic and suspected as the causative agent of synaptic dysfunction and neuronal loss in Alzheimer's disease (AD). Previously, we have shown that a small, D-amino acid Aß42-oligomer interacting peptide (D-AIP) can neutralize human Aß42-mediated toxicity using in vitro and cell-based assays. In the present longitudinal study using a transgenic Drosophila melanogaster model, advanced live confocal imaging and mass spectrometry imaging (MALDI-MSI) showed that the eight amino acid D-AIP can attenuate Aß42-induced toxicity in vivo. By separating male and female flies into distinct groups, the resultant distribution of ingested D-AIP was different between the sexes. The Aß42-induced 'rough eye' phenotype could be rescued in the female transgenics, likely because of the co-localization of D-AIP with human Aß42 in the female fly heads. Interestingly, the phenotype could not be rescued in the male transgenics, likely because of the co-localization of D-AIP with a confounding male-specific sex peptide (Acp70A candidate in MSI spectra) in the gut of the male flies. As a novel, more cost-effective strategy to prevent toxic amyloid formation during the early stages of AD (i.e. neutralization of toxic low-order Aß42 oligomers without creating larger aggregates in the process), our longitudinal study establishes that D-AIP is a stable and highly effective neutralizer of toxic Aß42 peptides in vivo. Cover Image for this issue: doi: 10.1111/jnc.14512.

Structural Analysis and Inhibition of TraE from the pKM101 Type IV Secretion System.

Gram-negative bacteria use type IV secretion systems (T4SSs) for a variety of macromolecular transport processes that include the exchange of genetic material. The pKM101 plasmid encodes a T4SS similar to the well-studied model systems from Agrobacterium tumefaciens and Brucella suis Here, we studied the structure and function of TraE, a homolog of VirB8 that is an essential component of all T4SSs. Analysis by X-ray crystallography revealed a structure that is similar to other VirB8 homologs but displayed an altered dimerization interface. The dimerization interface observed in the X-ray structure was corroborated using the bacterial two-hybrid assay, biochemical characterization of the purified protein, and in vivo complementation, demonstrating that there are different modes of dimerization among VirB8 homologs. Analysis of interactions using the bacterial two-hybrid and cross-linking assays showed that TraE and its homologs from Agrobacterium, Brucella, and Helicobacter pylori form heterodimers. They also interact with heterologous VirB10 proteins, indicating a significant degree of plasticity in the protein-protein interactions of VirB8-like proteins. To further assess common features of VirB8-like proteins, we tested a series of small molecules derived from inhibitors of Brucella VirB8 dimerization. These molecules bound to TraE in vitro, docking predicted that they bind to a structurally conserved surface groove of the protein, and some of them inhibited pKM101 plasmid transfer. VirB8-like proteins thus share functionally important sites, and these can be exploited for the design of specific inhibitors of T4SS function.

Development of DNA Nanostructures for High-Affinity Binding to Human Serum Albumin.

The development of nucleic acid therapeutics has been hampered by issues associated with their stability and in vivo delivery. To address these challenges, we describe a new strategy to engineer DNA structures with strong binding affinity to human serum albumin (HSA). HSA is the most abundant protein in the blood and has a long circulation half-life (19 days). It has been shown to hinder phagocytosis, is retained in tumors, and aids in cellular penetration. Indeed, HSA has already been successfully used for the delivery of small-molecule drugs and nanoparticles. We show that conjugating dendritic alkyl chains to DNA creates amphiphiles that exhibit high-affinity (Kd in low nanomolar range) binding to HSA. Notably, complexation with HSA did not hinder the activity of silencing oligonucleotides inside cells, and the degradation of DNA strands in serum was significantly slowed. We also show that, in a site-specific manner, altering the number and orientation of the amphiphilic ligand on a self-assembled DNA nanocube can modulate the affinity of the DNA cage to HSA. Moreover, the serum half-life of the amphiphile bound to the cage and the protein was shown to reach up to 22 hours, whereas unconjugated single-stranded DNA was degraded within minutes. Therefore, adding protein-specific binding domains to DNA nanostructures can be used to rationally control the interface between synthetic nanostructures and biological systems. A major challenge with nanoparticles delivery is the quick formation of a protein corona (i.e., protein adsorbed on the nanoparticle surface) upon injection to biological media. We foresee such DNA cage-protein complexes as new tools to study the role of this protein adsorption layer with important implications in the efficient delivery of RNAi therapeutics in vitro and in vivo.

Soluble CD109 binds TGF-ß and antagonizes TGF-ß signalling and responses.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine implicated in many diseases, including tissue fibrosis and cancer. TGF-ß mediates diverse biological responses by signalling through type I and II TGF-ß receptors (TßRI and TßRII). We have previously identified CD109, a glycosylphosphatidylinositol (GPI)-anchored protein, as a novel TGF-ß co-receptor that negatively regulates TGF-ß signalling and responses and demonstrated that membrane-anchored CD109 promotes TGF-ß receptor degradation via a SMAD7/Smurf2-mediated mechanism. To determine whether CD109 released from the cell surface (soluble CD109 or sCD109) also acts as a TGF-ß antagonist, we determined the efficacy of recombinant sCD109 to interact with TGF-ß and inhibit TGF-ß signalling and responses. Our results demonstrate that sCD109 binds TGF-ß with high affinity as determined by surface plasmon resonance (SPR) and cell-based radioligand binding and affinity labelling competition assays. SPR detected slow dissociation kinetics between sCD109 and TGF-ß at low concentrations, indicating a stable and effective interaction. In addition, sCD109 antagonizes TGF-ß-induced Smad2/3 phosphorylation, transcription and cell migration. Together, our results suggest that sCD109 can bind TGF-ß, inhibit TGF-ß binding to its receptors and decrease TGF-ß signalling and TGF-ß-induced cellular responses.

Novel zinc-binding site in the E2 domain regulates amyloid precursor-like protein 1 (APLP1) oligomerization.

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438-452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.

An immunogenic peptide in the A-box of HMGB1 protein reverses apoptosis-induced tolerance through RAGE receptor.

Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.

Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4.

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B(12) to the cytoplasm, impairing the vitamin B(12)-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B(12) transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B(12) transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B(12)-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B(12) to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.

Leucine-responsive regulatory protein Lrp and PapI homologues influence phase variation of CS31A fimbriae.

CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell.

Unveiling the Molecular Mechanisms of the Type-IX Secretion System's Response Regulator: Structural and Functional Insights.

The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo . First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX's effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX's dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae , but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

A platinum(II) phenylphenanthroimidazole with an extended side-chain exhibits slow dissociation from a c-Kit G-quadruplex motif.

A series of three platinum(II) phenanthroimidazoles each containing a protonable side-chain appended from the phenyl moiety through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) were evaluated for their capacities to bind to human telomere, c-Myc, and c-Kit derived G-quadruplexes. The side-chain has been optimized to enable a multivalent binding mode to G-quadruplex motifs, which would potentially result in selective targeting. Molecular modeling, high-throughput fluorescence intercalator displacement (HT-FID) assays, and surface plasmon resonance (SPR) studies demonstrate that complex 2 exhibits significantly slower dissociation rates compared to platinum phenanthroimidazoles without side-chains and other reported G-quadruplex binders. Complex 2 showed little cytotoxicity in HeLa and A172 cancer cell lines, consistent with the fact that it does not follow a telomere-targeting pathway. Preliminary mRNA analysis shows that 2 specifically interacts with the ckit promoter region. Overall, this study validates 2 as a useful molecular probe for c-Kit related cancer pathways.

Binding between insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 is not influenced by glucose or 2-deoxy-D-glucose.

A recent report (Zhong, D., Xiong, L., Liu, T., Liu, X., Liu, X., Chen, J., Sun, S. Y., Khuri, F. R., Zong, Y., Zhou, Q., and Zhou, W. (2009) J. Biol. Chem. 284, 23225-23233) details that 2-deoxy-D-glucose (2-DG), a well known inhibitor of glycolysis and a candidate antineoplastic agent, also induces insulin-like growth factor 1 receptor (IGF-1R) signaling through the inhibition of insulin-like growth factor 1-insulin-like growth factor-binding protein 3 (IGF-1-IGFBP-3) complex formation. Zhong et al. hypothesized that disrupted IGF-1/IGFBP-3 binding by 2-DG led to increased free IGF-1 concentrations and, consequently, activation of IGF-1R downstream pathways. Because their report suggests unprecedented off-target effects of 2-DG, this has profound implications for the fields of metabolism and oncology. Using ELISA, surface plasmon resonance, and novel "intensity-fading" mass spectrometry, we now provide a detailed characterization of complex formation between IGF-1 and IGFBP-3. All three of these independent methods demonstrated that there was no effect of glucose or 2-DG on the interaction between IGF-1 and IGFBP-3. Furthermore, we show examples of 2-DG exposure associated with reduced rather than increased IGF-1R and AKT activation, providing further evidence against a 2-DG increase in IGF-1R activation by IGF-1-IGFBP-3 complex disruption.

Lrp-DNA complex stability determines the level of ON cells in type P fimbriae phase variation.

F165(1) and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors that play a key role in the establishment of disease caused by extraintestinal Escherichia coli (ExPEC) strains. They are both under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Our present study demonstrates that the high level of ON cells found during F165(1) phase variation is due to altered stability of the DNA complex formed by the leucine-responsive regulatory protein (Lrp) at its repressor binding sites 1-3; after each cell cycle, complex formation is also modulated by the local regulator FooI (homologue to PapI) which promotes the transit of Lrp towards its activator binding sites 4-6. Furthermore, we identified two nucleotides (T490, G508) surrounding the Lrp binding site 1 that are critical to maintaining a high OFF to ON switch rate during F165(1) phase variation, as well as switching Pap fimbriae towards the OFF state.

Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B12 metabolism.

The genes MMACHC and MMADHC encode critical proteins involved in the intracellular metabolism of cobalamin. Two clinical features, homocystinuria and methylmalonic aciduria, define inborn errors of these genes. Based on disease phenotypes, MMADHC acts at a branch point for cobalamin delivery, apparently exerting its function through interaction with MMACHC that demonstrates dealkylase and decyanase activities. Here we present biophysical analyses of MMADHC to identify structural features and to further characterize its interaction with MMACHC. Two recombinant tag-less isoforms of MMADHC (MMADHCΔ1-12 and MMADHCΔ1-61) were expressed and purified. Full length MMACHC and full length MMADHC were detected in whole cell lysates of human cells; by Western blotting, their molecular masses corresponded to purified recombinant proteins. By clear-native PAGE and by dynamic light scattering, recombinant MMADHCs were stable and monodisperse. Both species were monomeric, adopting extended conformations in solution. Circular dichroism and secondary structure predictions correlated with significant regions of disorder within the N-terminal domain of MMADHC. We found no evidence that MMADHC binds cobalamin. Phage panning against MMADHC predicted four binding regions on MMACHC, two of which overlap with predicted sites on MMACHC at which it may self-associate. Specific, concentration-dependent responses were observed for MMACHC binding to itself and to both MMADHC constructs. As estimated in the sub-micromolar range, the binding of MMACHC to itself was weaker compared to its interaction with either of the MMADHC isoforms. We propose that the function of MMADHC is exerted through its structured C-terminal domain via interactions with MMACHC.

Biophysical characterization as a tool to predict amyloidogenic and toxic properties of amyloid-ß42 peptides.

Amyloid-ß42 (Aß42) peptides are central to the amyloid pathology in Alzheimer's disease (AD). As biological mimetics, properties of synthetic Aß peptides usually vary between vendors and batches, thus impacting the reproducibility of experimental studies. Here, we tested recombinantly expressed Aß42 (Asp1 to Ala42) against synthetic Aß42 from different suppliers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), circular dichroism (CD) spectroscopy, thioflavin T aggregation, surface plasmon resonance, and MTT cell viability assays. Overall, our recombinant Aß42 provided a reproducible mimetic of desired properties. Across experimental approaches, the combined detection of Aß42 dimers and random coil to ß-sheet transition only correlated with aggregation-prone and cytotoxic peptides. Conclusively, combining MALDI-MS with CD appears to provide a rapid, reliable means to predict the 'bioactivity' of Aß42.