Monomer Binding Research Articles

De novo aggregation of Alzheimer\u2019s A\u03b225-35 peptides in a lipid bilayer

A potential mechanism of cytotoxicity attributed to Alzheimer’s Aβ peptides postulates that their aggregation disrupts membrane structure causing uncontrollable permeation of Ca2+ ions. To gain molecular insights into these processes, we have performed all-atom explicit solvent replica exchange with solute tempering molecular dynamics simulations probing aggregation of the naturally occurring Aβ fragment Aβ25-35 within the DMPC lipid bilayer. To compare the impact produced on the bilayer by Aβ25-35 oligomers and monomers, we used as a control our previous simulations, which explored binding of Aβ25-35 monomers to the same bilayer. We found that compared to monomeric species aggregation results in much deeper insertion of Aβ25-35 peptides into the bilayer hydrophobic core causing more pronounced disruption in its structure. Aβ25-35 peptides aggregate by incorporating monomer-like structures with stable C-terminal helix. As a result the Aβ25-35 dimer features unusual helix head-to-tail topology supported by a parallel off-registry interface. Such topology affords further growth of an aggregate by recruiting additional peptides. Free energy landscape reveals that inserted dimers represent the dominant equilibrium state augmented by two metastable states associated with surface bound dimers and inserted monomers. Using the free energy landscape we propose the pathway of Aβ25-35 binding, aggregation, and insertion into the lipid bilayer.

Recruiting monomer for dimer formation: resolving the antagonistic mechanisms of novel immune check point inhibitors against Programmed Death Ligand-1 in cancer immunotherapy

ABSTRACTThe design of small molecule antagonists against Programmed Death Ligand-1 (PD-L1) has been the recent highlight of the immune checkpoint blockade therapy. This interventive approach has been potentiated by the development of BMS compounds; BMS-1001 and BMS-1166, which exert their therapeutic activities by inducing dimerisation of PD-L1; a molecular mechanism that has remained unclear. For the first time, we resolve the dynamical events that underlie the antagonistic mechanisms of BMS-1001 and BMS-1166 when bound to PD-L1 using an all-atom molecular dynamics (MD) simulations approach and free binding energy Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) calculations. Time-scale dynamical findings revealed that upon binding a PD-L1 monomer, the BMS-compounds gradually facilitated the ‘inbound’ motion of another PD-L1 monomer in the same conformational phase space up till dimer formation. Moreover, the non-liganded PD-L1 monomer exhibited the highest structural flexibility and atomistic motions relative to the BMS-liganded monomer as revealed by post-MD trajectory analyses using root mean square deviation (RMSD) and root mean square fluctuations (RMSF) parameters. Trajectory investigations into ligand motions also revealed that the BMS compounds exhibited mechanistic transitions from the monomeric binding site (monomer A) where they were initially bound, to the second monomeric site (monomer B) where they were strongly bound, followed by eventual high-affinity interactions at the tunnel-like binding cleft formed upon the dimerisation of both PD-L1 monomers. These findings present a model that describes the mechanism by which the BMS compounds induce PD-L1 dimerisation and could further enhance the design of highly selective and novel monomeric recruiters of PD-L1 in cancer immunotherapy.

Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/β3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.

Tailoring Biomolecular Interactions of Hybrid Nanostructures for their Diagnostic and Therapeutic Applications in Neurodegenerative Diseases

Alzheimer and Parkinson disease are common neurodegenerative disorders characterized by progressive structural and functional loss of the central nervous system. The foremost neuropathological events involve oxidative stress, mitochondrial dysfunction, inflammation and apoptosis. The disease-specific proteinopathies consists soluble aggregates including oligomers and protofibrils along with their phosphorylated variants deposited as plaque. The dynamic adsorption of these proteins on nanoparticle surface bestows with different physicochemical features and elicits the biological response. We show, in our approach molecular chemistry and optical characteristics of curcumin were used to elucidate the lanthanum carbonate nanospheres (REM100) and protein interactions via spontaneous binding of Aβ monomer and Aβ aggregates on Curcumin-REM100. The Protein concentration-dependent steady-state fluorescence enhancement of the curcumin and Langmuir protein adsorption model elucidates the maximum surface coverage and an apparent dissociation constant. Based on all experimental outcomes we demonstrated curcumin-REM100 as an efficient fluorescent probe able to delineate the monomer and the aggregate state of Amyloid-β with the Limit of detection (LOD) of 0.2 ±0.045 μM, the value is 30 times more sensitive than conventional Thioflavin-T (ThT) fluorescence emission intensity. The therapeutic approach uses the melatonin enriched polydopamine nanostructures for triggering robust neuroprotection against rotenone-induced neuroblastoma PD model. This causes reduction in cellular ROS level, re-establishing mitochondrial membrane potential, inhibiting caspase-dependent and independent apoptosis pathway and reducing alpha-synuclein phosphorylation at serine 129. The further in-vitro and organotypic mice brain slice culture-based therapeutic assessments have shown the superior neuroprotective potential of the developed nanoformulation. Considering the emerging interest in development of hybrid nanoformulation, our approaches elucidated diagnostic and therapeutic interventions in common neurodegenerative diseases.

Single-Stranded DNA Binding and Crosslinking Activities of the Viral Restriction Factor Apobec3G Characterized by Force Spectroscopy

APOBEC3G (A3G) is a human deaminase able to inhibit human immunodeficiency virus type 1 (HIV-1). A3G reduces HIV-1 infectivity via two independent mechanisms, including enzymatic hyper-mutation of the viral genome, and non-enzymatic stalling of the reverse transcription. However, the physical mechanism of these A3G activities remain unclear. Here we use force spectroscopy to show that the ssDNA-bound A3G exists in the rapid (1-10 s) conformational equilibrium between its “globular” state with its NTD and CTD bound to each other, with free energy ∼2.4 kBT, and its extended “dumbbell” state with its domains unbound, but connected by ∼0.6 nm flexible 7 aa linker. Low free energy and fast kinetics of this conformational transition in ssDNA-bound A3G suggest that both of these states are functionally important. We also measure much slower rates of monomeric A3G binding to and unbinding from ssDNA, which are of 107 M−1.s−1 and 0.014 s−1, respectively. However, while all of dimerization deficient FW A3G mutant dissociates with that rate, the a majority of the WT A3G oligomerizes upon binding within 10 s, and does not dissociate for over 10 min. We show that the A3G monomers can crosslink two DNA strands, leading to major construct shortening. For the WT A3G oligomerized on ssDNA the strand crosslinking process takes ∼300 s, while it only takes ∼10 s for the FW A3G mutant. In both cases crosslinking is rate-limited by one of the A3G domain unbinding one ssDNA strand and re-binding another strand. Taken together with other published data, our results suggest that the ssDNA-crosslinking A3G monomers are calalyticalycatalytically active, while the oligomerized A3Gs are catalytically inactive, and present a reverse transcription road block.

Multiple Interactions of the Oct-1 (POU2F1) Transcription Factor with PORE and MORE Sites

The Oct-1 (POU2F1) transcription factor is one of the most important regulatory proteins in humans and other mammals. An increase in Oct-1 aids the resistance to oxidative and cytotoxic stresses and radiation exposure. A high level of Oct-1 is found in many human tumors and correlates with low survival. Oct-1 interacts with its binding sites as a monomer, a homodimer, or a multimer. The nucleotide sequence of the Oct-1 binding site determines the character of interaction and the conformation of Oct-1 on target DNA, thus influencing the binding of Oct-1 co-repressors and co-activators. Nucleotide substitutions were introduced in all positions of the PORE and MORE sequences and tested for effect on the Oct-1 capability of forming monomeric and dimeric DNA-protein complexes. The position and nature of nucleotide substitutions were found to affect the type of Oct-1 binding to DNA. Several substitutions suppressed the formation of dimers, while others stimulated the process. Certain nucleotide substitutions completely prevented the binding of both monomers and dimers. The Oct-1 concentration in the cell is another factor that affects the character of DNA-protein interactions. Based on the results, the nature and affinity of interaction with Oct-1 is possible to predict from the nucleotide sequence for PORE and MORE sites of the human genome.

Palladium Acetate Trimer: Understanding Its Ligand-Induced Dissociation Thermochemistry Using Isothermal Titration Calorimetry, X-ray Absorption Fine Structure, and 31P Nuclear Magnetic Resonance

Palladium acetate trimer, Pd3(OAc)6, is a common precursor for Pd nanoparticle synthesis and an important precatalyst for homogeneous catalysis reactions. Added ligands (L, e.g., phosphines) are well-known to dissociate the Pd3(OAc)6 trimer into Pd(OAc)2 monomers and form Pd(OAc)2(L)2 complexes. Despite the importance of the trimer dissociation and ligand–monomer binding thermodynamics on the properties of the resulting Pd complex, little is known about either reaction. Using a combination of isothermal titration calorimetry (ITC), X-ray absorption fine structure (XAFS), and 31P nuclear magnetic resonance (31P NMR) on trimer samples containing monomer impurities, we developed a methodology and a trimer–monomer ITC model to obtain the Gibbs free energy, enthalpy, and entropy for both reactions in toluene and quantify the monomer content. The results provide the following, previously inaccessible, quantitative insights on the reactions. Trioctylphosphine (TOP)-monomer binding reaction is enthalpically driven (ΔH = −187 ± 8 kJ/mol), but the entropy loss due to binding (−TΔS = 84 ± 8 kJ/mol) was significant. However, while the trimer dissociation is enthalpically uphill (ΔH = 323 ± 35 kJ/mol), the increase in entropy due to the formation of monomers was very large (−TΔS = −266 ± 35 kJ/mol). The entropy gains and losses for both reactions have major contributions to each reaction Gibbs free energy and almost compensate for each other, making the overall reaction (i.e., Pd3(OAc)6 + 6 TOP ↔ 3 Pd(OAc)2(TOP)2) enthalpically driven. The results show the importance of determining the relative enthalpy and entropy contributions to the free energies for the complete thermochemical cycle of Pd acetate trimer dissociation and reaction with TOP. The methodology can be generalized to other ligands and metal or nonmetal homopolymers to obtain detailed thermochemistry of thermodynamically or kinetically unfavorable reactions.

Resolution of the Holliday junction recombination intermediate by human GEN1 at the single-molecule level.

Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ) after the dissolution of the nuclear membrane. GEN1 dimerization has been suggested to play key role in the resolution of the HJ, but the kinetic details of its reaction remained elusive. Here, single-molecule FRET shows how human GEN1 binds the HJ and always ensures its resolution within the lifetime of the GEN1-HJ complex. GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. GEN1 monomer remains tightly bound with no apparent dissociation until GEN1 dimer is formed and the HJ is fully resolved. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction. Furthermore, GEN1 monomer binds singly-cleaved HJ tighter than intact HJ providing a fail-safe mechanism if GEN1 dimer or one of its monomers dissociates after the first cleavage. The tight binding of GEN1 monomer to intact- and singly-cleaved HJ empowers it as the last resort to process HJs that escape the primary mechanisms.

Thermoplastic Overmolding onto Injection-Molded and In Situ Polymerization-Based Polyamides.

We investigated products manufactured by in situ polymerization, which were reinforced with overmolded ribs. We developed our own mold and prototype product for the project. We used three different materials as preform: a material with a magnesium catalyst, manufactured by in situ polymerization, a Brüggemann AP-NYLON-based in situ polymerization material and an injection-molded PA6 (Durethan B30S, Lanxess GmbH) material. The ribs were formed from the same PA6 material (Durethan B30S, Lanxess GmbH). We examined the effect of the different technological parameters through the pull-off of the overmolded ribs. We measured the effect of melt temperature, holding pressure and holding time, and mold temperature. Considering the individual preforms, we pointed out that monomer migration and binding strength are related, which we concluded from the temperature-dependent mass loss of the materials, measured by thermogravimetric analysis (TGA). Finally, we designed a mold suitable for manufacturing overmolded parts. We designed and built pressure and temperature sensors into the mold to examine and analyze pressures and temperatures around the welding zone of the materials.

Self-Assembly of Nanocubic Molecular Capsules via Solvent-Guided Formation of Rectangular Blocks.

We investigate the mechanism underlying the self-assembly of gear-shaped amphiphilic molecules into a highly ordered nanocubic capsule ("nanocube") in aqueous methanol. Simulation results show that the solvent molecules play a significant role in the assembly process by directing the primitive intermediates to orthogonal/rectangular shapes, thus creating appropriate building blocks for cubic assembly while avoiding off-pathway stacked aggregates. Free-energy analyses reveal that the interplay of the direct intermonomer interaction and the solvent-mediated repulsion between large aromatic cores (via preferential solvation of methanol on hydrophobic surfaces) leads to the strong trend for perpendicular binding of monomers and hence the solvent-guided formation of rectangular blocks. Furthermore, we report the self-assembly simulation of the nanocube using replica exchange with solute tempering and demonstrate that the simulation can predict a highly ordered nanocapsule structure, assembly intermediates, and encapsulated molecules, which helps promote computer-aided design of functional molecular self-assemblies in explicit solvent.

Vibrio cholerae chromosome 2 copy number is controlled by the methylation-independent binding of its monomeric initiator to the chromosome 1 crtS site.

Bacteria contain a primary chromosome and, frequently, either essential secondary chromosomes or dispensable megaplasmids of plasmid origin. Incoming plasmids are often poorly adapted to their hosts and their stabilization requires integration with the host's cellular mechanisms in a process termed domestication. All Vibrio, including pathogenic species, carry a domesticated secondary chromosome (Chr2) where replication is coordinated with that of the primary chromosome (Chr1). Chr2 replication is triggered by the replication of an intergenic sequence (crtS) located on Chr1. Yet, the molecular mechanisms by which crtS replication controls the initiation of Chr2 replication are still largely unknown. In this study, we show that crtS not only regulates the timing of Chr2 initiation but also controls Chr2 copy number. We observed and characterized the direct binding of the Chr2 initiator (RctB) on crtS. RctB binding to crtS is independent of its methylation state. RctB molecules, which naturally form dimers, preferentially bind to crtS as monomers, with DnaK/J protein chaperones shown to stimulate binding of additional RctB monomers on crtS. In this study, we addressed various hypothesis of how replication of crtS could trigger Chr2 replication and provide new insights into its mode of action.

A massively parallel reporter assay reveals context-dependent activity of homeodomain binding sites in vivo.

Cone-rod homeobox (CRX) is a paired-like homeodomain transcription factor (TF) and a master regulator of photoreceptor development in vertebrates. The in vitro DNA binding preferences of CRX have been described in detail, but the degree to which in vitro binding affinity is correlated with in vivo enhancer activity is not known. In addition, paired-class homeodomain TFs can bind DNA cooperatively as both homodimers and heterodimers at inverted TAAT half-sites separated by 2 or 3 nucleotides. This dimeric configuration is thought to mediate target specificity, but whether monomeric and dimeric sites encode distinct levels of activity is not known. Here, we used a massively parallel reporter assay to determine how local sequence context shapes the regulatory activity of CRX binding sites in mouse photoreceptors. We assayed inactivating mutations in more than 1700 TF binding sites and found that dimeric CRX binding sites act as stronger enhancers than monomeric CRX binding sites. Furthermore, the activity of dimeric half-sites is cooperative, dependent on a strict 3-bp spacing, and tuned by the identity of the spacer nucleotides. Saturating single-nucleotide mutagenesis of 195 CRX binding sites showed that, on average, changes in TF binding site affinity are correlated with changes in regulatory activity, but this relationship is obscured when considering mutations across multiple cis-regulatory elements (CREs). Taken together, these results demonstrate that the activity of CRX binding sites is highly dependent on sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain binding sites that may be conserved in other cell types.

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.

The first transmembrane region of complement component-9 acts as a brake on its self-assembly

Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.

Molecular model of J-aggregated pseudoisocyanine fibers.

Aggregated cyanines form ordered supramolecular structures with the potential to transport energy efficiently over long distances, a hallmark of photosynthetic light-harvesting complexes. In concentrated aqueous solution, pseudoisocyanine (PIC) spontaneously forms fibers with a chiral J-band red-shifted 1600 cm-1 from the monomeric 0-0 transition. A cryogenic transmission electron microscopy analysis of these fibers show an average fiber width of 2.89 nm, although the molecular-level structure of the aggregate is currently unknown. To determine a molecular model for these PIC fibers, the calculated spectra and dynamics using a Frenkel exciton model are compared to experiment. A chiral aggregate model in which the PIC monomers are neither parallel nor orthogonal to the long axis of the fiber is shown to replicate the experimental spectra most closely. This model can be physically realized by the sequential binding of PIC dimers and monomers to the ends of the fiber. These insights into the molecular aggregation model for aqueous PIC can also be applied to other similar cyanine-based supramolecular complexes with the potential for long-range energy transport, a key building block for the rational design of novel excitonic systems.

Chiral Monomers Ensure Orientational Specificity of Monomer Binding During Polymer Self-Replication.

Biomolecular homochirality is universally observed in living systems but the molecular and evolutionary dynamics that led to its emergence are unknown. In fact, there are significant disadvantages in using chiral monomers for polymerization, which include enantiomeric cross-inhibition in racemic medium and under-utilization of available resources for self-replication in the primordial environment. Nevertheless, most investigations of homochirality in living systems assume that the individual primordial monomers were chiral prior to the formation of self-replicating polymer and therefore focus on identifying a symmetry-breaking mechanism that might choose one enantiomer over the other in a racemic medium. Within the premise that the extant biomolecules are products of molecular evolution, we ask a related but distinct question: why is an achiral monomer molecule disfavored? Here we identify an evolutionary advantage for molecular evolution to choose chiral over achiral monomers to construct primordial self-replicating polymers. We argue that when polymerization is constrained to proceed in only one direction along the template, as in DNA, evolution favors chiral monomers and homochiral polymers. This evolutionary advantage stems from the ability of a chiral monomer to bond with the template in only one orientation relative to the template monomer, along the direction of polymerization. An achiral monomer, on the other hand, offers more than one possible orientation for bonding with the template monomer, due to the presence of symmetry elements in its structure, which would lead to inhibition of polymerization. We show that the requirement of orientational specificity leads to monomer chirality, by using a known relationship between rotational and reflection symmetry elements, within the constraint that the resultant polymers are helical.

IL-3 but not monomeric IgE regulates Fc\u03b5RI levels and cell survival in primary human basophils

Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

ABSTRACTThe biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM. A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM. The expression of this gene is controlled by the Streptomyces antibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis in Streptomyces and open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis by Streptomyces natalensis could be extrapolated to the production of different compounds by other species.

Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response

The innate RNA sensor RIG-I is critical in the initiation of antiviral type I interferons (IFNs) production upon recognition of "non-self" viral RNAs. Here, we identify a host-derived, IFN-inducible long noncoding RNA, lnc-Lsm3b, that can compete with viral RNAs in the binding of RIG-I monomers and feedback inactivate the RIG-I innate function at late stage of innate response. Mechanistically, binding of lnc-Lsm3b restricts RIG-I protein's conformational shift and prevents downstream signaling, thereby terminating type I IFNs production. Multivalent structural motifs and long-stem structure arecritical features of lnc-Lsm3b for RIG-I binding and inhibition. These data reveal a non-canonical self-recognition mode in the regulation of immune response and demonstrate an important role of an inducible "self" lncRNA acting as a potent molecular decoy actively saturating RIG-I binding sites to restrict the duration of "non-self" RNA-induced innate immune response and maintaining immune homeostasis, with potential utility in inflammatory disease management.

Identification of distinct conformations associated with monomers and fibril assemblies of mutant huntingtin.

The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1-4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT.