Pleomorphic Ensembles: Formation of Large Clusters Composed of Weakly Interacting Multivalent Molecules

Molecular interactions of importance to cell biology are subject to sol-gel transitions: large clusters of weakly interacting multivalent molecules (gel phase) are produced at a critical concentration of monomers. Examples include cell-cell and cell-matrix adhesions, nucleoprotein bodies, and cell signaling platforms. We use the term pleomorphic ensembles (PEs) to describe these clusters, because they have dynamic compositions and sizes and have rapid turnover of their molecular constituents; this plasticity can be highly responsive to cellular signals. The classical polymer physical chemistry theory developed by Flory and Stockmayer provides a brilliant framework for treating multivalent interactions for simple idealized systems. But the complexity and variability of PEs challenges existing modeling approaches. Here we describe and validate a computational algorithm that extends the Flory-Stockmayer formalism to overcome the limitations of analytic theories. We divide the problem by deterministically calculating the fraction of bound sites for each type of binding site, followed by the stochastic assignment of the bonds to a finite number of molecules. The method allows for high valency within many different kinds of interacting molecules and site types, permits simulation of steady-state distributions, as well as assembly kinetics, and can treat cooperative binding within one of the interacting molecules. We then apply our method to the analysis of interactions in the nephrin-Nck-N-Wasp signaling system, demonstrating how multivalent layered scaffolds produce PEs at low monomer concentrations despite weak binding interactions. We show how the experimental data for this system are most consistent with synergistic cooperative interactions between Nck and N-Wasp.     

Pleomorphic Ensembles: Formation of Large Clusters Composed of Weakly Interacting Multivalent Molecules

Molecular interactions of importance to cell biology are subject to sol-gel transitions: large clusters of weakly interacting multivalent molecules (gel phase) are produced at a critical concentration of monomers. Examples include cell-cell and cell-matrix adhesions, nucleoprotein bodies, and cell signaling platforms. We use the term pleomorphic ensembles (PEs) to describe these clusters, because they have dynamic compositions and sizes and have rapid turnover of their molecular constituents; this plasticity can be highly responsive to cellular signals. The classical polymer physical chemistry theory developed by Flory and Stockmayer provides a brilliant framework for treating multivalent interactions for simple idealized systems. But the complexity and variability of PEs challenges existing modeling approaches. Here we describe and validate a computational algorithm that extends the Flory-Stockmayer formalism to overcome the limitations of analytic theories. We divide the problem by deterministically calculating the fraction of bound sites for each type of binding site, followed by the stochastic assignment of the bonds to a finite number of molecules. The method allows for high valency within many different kinds of interacting molecules and site types, permits simulation of steady-state distributions, as well as assembly kinetics, and can treat cooperative binding within one of the interacting molecules. We then apply our method to the analysis of interactions in the nephrin-Nck-N-Wasp signaling system, demonstrating how multivalent layered scaffolds produce PEs at low monomer concentrations despite weak binding interactions. We show how the experimental data for this system are most consistent with synergistic cooperative interactions between Nck and N-Wasp.     

Cooperating in the face of uncertainty: a consistent framework for understanding the evolution of cooperation

The evolution of cooperative behaviour, whereby individuals enhance the fitness of others at an apparent cost to themselves, represents one of the greatest paradoxes of evolution. Individuals that engage in such cooperative behaviour can, however, be favoured by natural selection if cooperative actions confer higher fitness than alternative actions. To understand the evolution of cooperative behaviour, the direct and indirect genetic benefits that individuals accrue in the present and future must be summed - this can be accomplished without any reference to the colorful vocabulary typically associated with studies of cooperation. When benefits are accrued indirectly through relatives or directly in the future individuals must be able to assess and enhance their probability of accruing those benefits and behave accordingly. We suggest that, in the same way that studies of kin recognition systems improved our understanding of how individuals assess and enhance their probability of accruing indirect benefits, studies of various forms of inheritance and reciprocation recognition systems will improve our understanding of how individuals assess and enhance their probability of accruing future benefits. Recognizing the parallel between studies of indirect fitness and future fitness, at multiple levels of analysis, will move us toward a simpler and more consistent framework for understanding the evolution of cooperative behaviour.     

Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy

Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity.     

Who's In and Who's Out—Compositional Control of Biomolecular Condensates

Biomolecular condensates are two- and three-dimensional compartments in eukaryotic cells that concentrate specific collections of molecules without an encapsulating membrane. Many condensates behave as dynamic liquids and appear to form through liquid–liquid phase separation driven by weak, multivalent interactions between macromolecules. In this review, we discuss current models and data regarding the control of condensate composition, and we describe our current understanding of the composition of representative condensates including PML nuclear bodies, P-bodies, stress granules, the nucleolus, and two-dimensional membrane localized LAT and nephrin clusters. Specific interactions, such as interactions between modular binding domains, weaker interactions between intrinsically disorder regions and nucleic acid base pairing, and nonspecific interactions, such as electrostatic interactions and hydrophobic interactions, influence condensate composition. Understanding how specific condensate composition is determined is essential to understanding condensates as biochemical entities and ultimately discerning their cellular and organismic functions.     

Confounding social and mating systems predictably lead to biased results when examining the evolution of cooperative breeding in cichlids: A response to Tanaka et al.

In 2017, we demonstrated that transitions to cooperative breeding in Lamprologine cichlid fishes were not related to a species’ social mating system (Dey et al. 2017. Nature Ecology & Evolution, 1 , 137). This contrasted previous evidence that monogamy (and a low degree of promiscuity) promoted transitions to cooperative breeding in other taxa. Recently, Tanaka et al. (2018. Ethology, 124 , 777–789) critiqued our study and argued that a re‐analysis of the data shows transitions to cooperative breeding are promoted by non‐monogamous mating systems. Here, we show that Tanaka et al.'s critique contains numerous inaccuracies. In addition, we show that the results put forth by Tanaka et al. emerge only under the extreme scenario in which all cooperative breeding species are classified as non‐monogamous, which we argue arises because Tanaka et al. confound social systems and mating systems. While we agree that there is uncertainty regarding the mating system of some Lamprologine species, we argue this uncertainty was sufficiently addressed through the extensive sensitivity analyses conducted in our original study.     

Reactivity of sulfhydryl group in peptides and poly-peptides with p-nitrophenylacetate. S to N acyl shift in cysteine.

The activity of poly (Cys-Ser-Phe-Glu-Glu) and related polypeptides as models of cysteine-proteases towards p-nitrophenylacetate was studied. The reaction leads to the formation of an S-acetylated form which proved to be quite stable, except at very high pH values. The presence of imidazole groups in the neighbourhood of sulfhydryl functions seems to result in an enhancement of the activity, which we attributed to a cooperative interaction. However, this interaction has no effect upon the S-deacylation rate. In the case of cysteine we found that the reaction with NPA occurred through a S to N acyl shift. Our results lead us to suggest that, unless convincing proofs of deacylation are given, the various models of cysteine-proteases studied so far do not behave as "true catalysts".     

Pseudosaccharide functionalized dendrimers as potent inhibitors of DC-SIGN dependent Ebola pseudotyped viral infection

The development of compounds with strong affinity for the receptor DC-SIGN is a topic of remarkable interest due to the role that this lectin plays in several pathogen infection processes and in the modulation of the immune response. DC-SIGN recognizes mannosylated and fucosylated oligosaccharides in a multivalent manner. Therefore, multivalent carbohydrate systems are required to interact in an efficient manner with this receptor and compete with the natural ligands. We have previously demonstrated that linear pseudodi- and pseudotrisaccharides are adequate ligands for DC-SIGN. In this work, we show that multivalent presentations of these glycomimetics based on polyester dendrons and dendrimers lead to very potent inhibitors (in the nanomolar range) of cell infection by Ebola pseudotyped viral particles by blocking DC-SIGN receptor. Furthermore, SPR model experiments confirm that the described multivalent glycomimetic compounds compete in a very efficient manner with polymannosylated ligands for binding to DC-SIGN.     

Cooperative breeding and monogamy in mammalian societies

Comparative studies of social insects and birds show that the evolution of cooperative and eusocial breeding systems has been confined to species where females mate completely or almost exclusively with a single male, indicating that high levels of average kinship between group members are necessary for the evolution of reproductive altruism. In this paper, we show that in mammals, the evolution of cooperative breeding has been restricted to socially monogamous species which currently represent 5 per cent of all mammalian species. Since extra-pair paternity is relatively uncommon in socially monogamous and cooperatively breeding mammals, our analyses support the suggestion that high levels of average kinship between group members have played an important role in the evolution of cooperative breeding in non-human mammals, as well as in birds and insects.     

Oxidative catalysts for the transformation of phenolic pollutants: a brief review

Phenolics are often produced as wastes by several industrial and agricultural activities. Many of these compounds and their derivatives are extremely dangerous to living organisms, because they are highly toxic and thus represent a serious environmental concern.

Conventional remediation methods of phenol-polluted systems have some disadvantages due to high cost, time-consuming procedures and formation of toxic residues. Conversely, the use of oxidative catalysts, both enzymatic or inorganic, is a promising alternative technology to address the clean up of such wastes. Oxidative enzymes and inorganic compounds, both naturally occurring in soil, behave as biotic and abiotic catalysts and support the transformation of phenolic compounds. The complete mineralization of phenolic pollutants as well as the formation of polymeric products, often less toxic than their precursors, may occur.

The present paper gives a brief review of many aspects concerning the properties of biotic and abiotic catalytic agents effective in the transformation of phenolic compounds. The main mechanisms of the processes as well as their feasibility for catalytic practical applications will be addressed. Examples of their potentiality in the detoxification of phenol-polluted systems will be provided, as well.     

Trivalent PEGylated platform for the conjugation of bioactive compounds

PEGylated multivalent structures are a new class of platform for biological applications due to their biocompatibility properties. Here, we present the synthesis of a trivalent structure 1 based on poly(ethylene glycol) units (PEG) as potential synthetic multifunctional carrier molecule. To evaluate whether this PEGylated platform could be useful for the conjugation of bioactive compounds, a well-known lipopolysaccharide (LPS) inhibitor 2, developed in our laboratory, was selected to be conjugated to 1. The LPS-neutralizing activity of the resulted conjugates and precursors was established using the chromogenic Limulus amebocyte lysate (LAL) assay. The trivalent structure 1 did not show LPS-binding activity, nonconjugate LPS inhibitor 2 showed high LPS-neutralizing activity, and the trivalent conjugate 4 displayed increased LPS-neutralizing activity and a reduced toxicity profile. These results prove the efficacy of this trivalent platform as a multivalent ligand scaffold for biological applications.     

Polyamino acids as synthetic enzymes: mechanism, applications and relevance to prebiotic catalysis

Polyamino acids, such as polyleucine, behave as synthetic enzymes in the asymmetric epoxidation of chalcone and other electron-deficient alkenes (the Julià-Colonna reaction). The influences of reaction conditions, of the molecular structure of the catalysts and of the scaling-up of the process on the enantioselectivity of the reaction have been determined. The kinetics and mechanism have been investigated using a soluble PEG-polyleucine conjugate, which behaves in a similar way to an enzyme, showing saturation kinetics for both chalcone and HOO-. Enantioselective catalysis is achieved with peptides with as few as five residues and scalemic catalysts show high chiral amplification. Here, we discuss the relevance of these-enzyme like catalysts to prebiotic processes, such as the role of small peptides in the formation of optically active cyanohydrins.     

Core and periphery functionalized dendrimers for transition metal catalysis; a covalent and a non-covalent approach

Dendrimers are well-defined hyperbranched macromolecules with characteristic globular structures for the larger systems. The recent impressive strides in synthetic procedures increased the accessibility of functionalized dendrimers at a practicable scale, resulting in a rapid development of dendrimer chemistry. Dendrimers have inspired many chemists to develop new materials and several applications have been explored, catalysis being one of them. The position of the catalytic site(s) as well as the spatial separation of the catalysts within the dendritic framework is of crucial importance. Dendrimers that are functionalized with transition metals in the core can potentially mimic properties of enzymes, their efficient natural counterparts, whereas the surface-functionalized systems have been proposed to fill the gap between homogeneous and heterogeneous catalysis. We prepared both core- and periphery-functionalized dendritic catalysts that are sufficiently large to enable separation by modern nanofiltration techniques. Here we review our recent findings using these promising novel transition metal-functionalized dendrimers as catalysts in several reactions. We will discuss some of the consequences of the architecturally different systems that have been studied and will elaborate on a novel non-covalent strategy of dendrimer functionalization.     

Influence of ligand valency on ligand-influenced monomer-dimer equilibrium systems and biological control mechanisms

Two specific ligand-influenced monomer-dimer equilibrium systems are discussed. Each has a ligand-to-dimer subunit ratio of 0.5. Equilibrium characteristics of the system are described in terms of the effect of a bivalent ligand on both experimental and theoretical analysis. It is shown that Hill expressions need to be modified for multivalent ligand systems, and care is needed in equilibrium parameter determination. Some unique properties of these systems are retained, yet others are altered in the presence of a multivalent ligand. A suggestion as to the minimum amount of information needed to describe completely a ligand-influenced monomerdimer system is given. The estrogen-receptor system is presented as an attractive biological model for both theoretical and experimental study of ligand-influenced polymerizing systems and their role in cellular control requirements.     

Benevolent Characteristics Promote Cooperative Behaviour among Humans

Cooperation is fundamental to the evolution of human society. We regularly observe cooperative behaviour in everyday life and in controlled experiments with anonymous people, even though standard economic models predict that they should deviate from the collective interest and act so as to maximise their own individual payoff. However, there is typically heterogeneity across subjects: some may cooperate, while others may not. Since individual factors promoting cooperation could be used by institutions to indirectly prime cooperation, this heterogeneity raises the important question of who these cooperators are. We have conducted a series of experiments to study whether benevolence, defined as a unilateral act of paying a cost to increase the welfare of someone else beyond one''s own, is related to cooperation in a subsequent one-shot anonymous Prisoner''s dilemma. Contrary to the predictions of the widely used inequity aversion models, we find that benevolence does exist and a large majority of people behave this way. We also find benevolence to be correlated with cooperative behaviour. Finally, we show a causal link between benevolence and cooperation: priming people to think positively about benevolent behaviour makes them significantly more cooperative than priming them to think malevolently. Thus benevolent people exist and cooperate more.     

Chromosome pairing affinity and quadrivalent formation in polyploids: do segmental allopolyploids exist?

When polyploid hybrids with closely related genomes are propagated by selfing or sib-breeding, the meiotic behaviour will turn into essentially autopolyploid behaviour as soon as the affinity between the genomes is sufficient to permit occasional homoeologous pairing. An allopolyploid will only be formed when the initial differentiation is sufficient to completely prevent homoeologous pairing (in some cases enhanced by specific genes), or when segregational dysgenesis prevents transmission of recombined chromosomes. A new polyploid hybrid may be considered a segmental allopolyploid and may show reduced multivalent formation as a result of preferential pairing between the least differentiated genomes. An established polyploid is either an autopolyploid or an allopolyploid. In exceptional cases it is thinkable that a stable segmental allopolyploid arises, in which some sets of chromosomes are well differentiated and behave as in an allopolyploid, whereas other sets are not well differentiated and behave as in an autopolyploid. No clear cases have been found in the literature so far. Key words : chromosome, pairing affinity, quadrivalent frequency, segmental allopolyploidy.     

Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM₁ and GM₁-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM₁ positive cooperativity. Surprisingly, we demonstrated fucosyl-GM₁ has approximately 7 times higher CTB binding capacity than GM₁. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM₁ caused the increased binding capacity. This was unintuitive, as GM₁ exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM₁ and GM₂, we observed the mild binding avidity fucosyl-GM₁ activated GM₂ receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM₂ receptors have negligible binding avidity in pure GM₂ bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.     

Multivalent carbocyanine molecular probes: synthesis and applications

Synergistic multivalent interactions can amplify desired chemical or biological molecular recognitions. We report a new class of multicarboxylate-containing carbocyanine dye constructs for use as optical scaffolds that not only serve as fluorescent antennas but also participate in structural assembly of the multivalent molecular construct. Three generations of carboxylate-terminating multivalent near-infrared carbocyanine probes from a dicarboxylic acid precursor dye (cypate) were prepared via its imino diacetic acid derivatives. Conjugation of the probes with D-(+)-glucosamine afforded dendritic arrays of the carbohydrates on an inner NIR chromophore core. All the multicarboxylate probes and their glucosamine conjugates have similar NIR spectral properties because conjugation occurred at distal positions to the inner chromophore core, thereby providing consistent and predictable spectral properties for their biological applications. Although light-induced photodamage equally affected the precursor dye, multicarboxylate probes, and their glucosamine derivatives, we observed that octacarboxylcypate (multivalent probe) was remarkably stable in different mediums at physiologically relevant temperatures relative to cypate, especially in basic mediums. Biodistribution studies in tumor-bearing nude mice show that all the glucosamine conjugates localized in the tumor but cypate was almost exclusively retained in the liver at 24 h postinjection. The tumor uptake does not correlate with the number of glucosamine tether on the multicarboxylate probe. Overall, the triglucosamine derivative appears to offer the best balance between high tumor uptake and low retention in nontarget tissues. These results suggest that multivalent molecular beacons are useful for assessing the beneficial effects of multivalency and for optimizing the biological and chemical properties of tissue-specific molecular probes.     

Emerging tools for synthetic genome design

Synthetic biology is an emerging discipline for designing and synthesizing predictable, measurable, controllable, and transformable biological systems. These newly designed biological systems have great potential for the development of cheaper drugs, green fuels, biodegradable plastics, and targeted cancer therapies over the coming years. Fortunately, our ability to quickly and accurately engineer biological systems that behave predictably has been dramatically expanded by significant advances in DNA-sequencing, DNA-synthesis, and DNA-editing technologies. Here, we review emerging technologies and methodologies in the field of building designed biological systems, and we discuss their future perspectives.