Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Protein SUMOylation and phase separation: partners in stress?

Protein SUMOylation is one of the most prevalent post-translational modifications (PTMs) and important for maintaining cellular homeostasis in response to various cellular stresses. Emerging evidence reveals the role of liquid-liquid phase separation (LLPS)/biomolecular condensates in cellular SUMOylation, potentially solving a puzzle regarding the cellular mechanism of SUMOylation regulation.

Get closer and make hotspots: liquid-liquid phase separation in plants.

Liquid-liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus-responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants.

A tetravalent peptide that binds to the RANK-binding region of TRAF6 via a multivalent interaction efficiently inhibits osteoclast differentiation.

Inhibition of osteoclast differentiation is a promising approach for the treatment of osteoporosis and rheumatoid arthritis. Receptor activator of nuclear factor kappa B (NF-κB) (RANK), which is an essential molecule for osteoclast differentiation, interacts with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to transduce downstream signals. Both RANK and TRAF6 have homo-trimeric structures, forming a multivalent interaction between the Pro-X-Glu-X-X-(aromatic/acidic) motif of RANK and the C-terminal domain of TRAF6 (TRAF-C), that markedly increases the binding affinity. Here, we designed a tetravalent peptide, RANK-tet, containing the TRAF-C-binding motif of RANK and found that RANK-tet binds to TRAF-C with high affinity. In contrast, a monomeric form of RANK-tet (RANK-mono) with the same TRAF-C-binding motif did not bind to TRAF-C, clearly indicating the multivalent interaction is strictly required for the high-affinity binding to TRAF-C. RANK-tet did not bind to a series of TRAF-C-mutants with an amino acid substitution in the RANK-binding region, indicating that RANK-tet specifically targets the RANK-binding region of TRAF-C. A cell-permeable form of RANK-tet that has poly-Arg residues at each C-terminal of the TRAF-C-binding motif efficiently inhibited the RANK ligand (RANKL)-induced differentiation of bone marrow cells to osteoclasts. Thus, this compound can be an effective anti-osteoclastogenic agent.

Multivalent butyrylcholinesterase inhibitor discovered by exploiting dynamic combinatorial chemistry.

In this study, we report the generation of a polymer-based dynamic combinatorial library (DCL) incorporating exchangeable side chains using acylhydrazone formation reaction. In combination with tetrameric butyrylcholinesterase (BChE), the most potent binding side chain was identified, and the information obtained was further used for the synthesis of a multivalent BChE inhibitor. In the in vitro biological evaluation, this multivalent inhibitor exhibited not only better inhibitory effect than the commercial reference but also high selectivity on BChE over acetylcholinesterase (AChE).

Identification and synthesis of an efficient multivalent E. coli heat labile toxin inhibitor __ A dynamic combinatorial chemistry approach.

A polymer based dynamic combinatorial library (DCL) was generated through condensation between aldehyde functionalized linear poly(glycidol) (APG) and galactose containing acylhydrazide derivatives. Pentameric E. coli heat labile enterotoxin B subunit (LTB) was subsequently applied to the DCL as external stimulus, resulting in amplification of a specific acylhydrazone side chain that was further used for the synthesis of a multivalent LTB inhibitor. In the in vitro biological evaluation, this inhibitor exhibited strong inhibition properties as well as low cytotoxicity.

Multivalent-Interaction-Driven Assembly of Discrete, Flexible, and Asymmetric Supramolecular Protein Nano-Prisms.

Current approaches to design monodisperse protein assemblies require rigid, tight, and symmetric interactions between oligomeric protein units. Herein, we introduce a new multivalent-interaction-driven assembly strategy that allows flexible, spaced, and asymmetric assembly between protein oligomers. We discovered that two polygonal protein oligomers (ranging from triangle to hexagon) dominantly form a discrete and stable two-layered protein prism nanostructure via multivalent interactions between fused binding pairs. We demonstrated that protein nano-prisms with long flexible peptide linkers (over 80 amino acids) between protein oligomer layers could be discretely formed. Oligomers with different structures could also be monodispersely assembled into two-layered but asymmetric protein nano-prisms. Furthermore, producing higher-order architectures with multiple oligomer layers, for example, 3-layered nano-prisms or nanotubes, was also feasible.

A Multivalent Structure-Specific RNA Binder with Extremely Stable Target Binding but Reduced Interaction with Nonspecific RNAs.

By greatly enhancing binding affinities against target biomolecules, multivalent interactions provide an attractive strategy for biosensing. However, there is also a major concern for increased binding to nonspecific targets by multivalent binding. A range of charge-engineered probes of a structure-specific RNA binding protein PAZ as well as multivalent forms of these PAZ probes were constructed by using diverse multivalent avidin proteins (2-mer, 4-mer, and 24-mer). Increased valency vastly enhanced the binding stability of PAZ to structured target RNA. Surprisingly, nonspecific RNA binding of multivalent PAZ can be reduced even below that of the PAZ monomer by controlling negative charges on both PAZ and multivalent avidin scaffolds. The optimized 24-meric PAZ showed nearly irreversible binding to target RNA with negligible binding to nonspecific RNA, and this ultra-specific 24-meric PAZ probe allowed SERS detection of intact microRNAs at an attomolar level.

Adaptive enzyme-responsive self-assembling multivalent apelin ligands for targeted myocardial infarction therapy.

Microvascular dysfunction following myocardial infarction exacerbates coronary flow obstruction and impairs the preservation of ventricular function. The apelinergic system, known for its pleiotropic effects on improving vascular function and repairing ischemic myocardium, has emerged as a promising therapeutic target for myocardial infarction. Despite its potential, the natural apelin peptide has an extremely short circulating half-life. Current apelin analogs have limited receptor binding efficacy and poor targeting, which restricts their clinical applications. In this study, we utilized an enzyme-responsive peptide self-assembly technique to develop an enzyme-responsive small molecule peptide that adapts to the expression levels of matrix metalloproteinases in myocardial infarction lesions. This peptide is engineered to respond to the high concentration of matrix metalloproteinases in the lesion area, allowing for precise and abundant presentation of the apelin motif. The changes in hydrophobicity allow the apelin motif to self-assemble into a supramolecular multivalent peptide ligand-SAMP. This self-assembly behavior not only prolongs the residence time of apelin in the myocardial infarction lesion but also enhances the receptor-ligand interaction through increased receptor binding affinity due to multivalency. Studies have demonstrated that SAMP significantly promotes angiogenesis after ischemia, reduces cardiomyocyte apoptosis, and improves cardiac function. This novel therapeutic strategy offers a new approach to restoring coronary microvascular function and improving damaged myocardium after myocardial infarction.

PD-L1 exosomes electrochemical sensor based on coordination of AgNCs and Zr4+: Multivalent peptide enhancing target capture efficiency and antifouling performance.

Programmed death ligand 1 (PD-L1) exosomes are important biomarkers of immune activation in the initial stages of treatment and can predict clinical responses to PD-1 blockade in various cancer patients. However, traditional PD-L1 exosome bioassays face challenges such as high interface fouling in complex detection environments, limited detection specificity, and poor clinical serum applicability. Inspired by the multi-branched structure of trees, a biomimetic tree-like multifunctional antifouling peptide (TMAP)-assisted electrochemical sensor was developed for high-sensitivity exosomes detection. Multivalent interaction of TMAP significantly enhances the binding affinity of PD-L1 exosomes, thanks to the designed branch antifouling sequence, TMAPs antifouling performance is further improved. The addition of Zr4+ forms coordination bonds with the exosome's lipid bilayer phosphate groups to achieve highly selective and stable binding without interference from protein activity. The specific coordination between AgNCs and Zr4+ contributes to a dramatic change in the electrochemical signals, and lowing detection limit. The designed electrochemical sensor exhibited excellent selectivity and a wide dynamic response within the PD-L1 exosome concentration range from 78 to 7.8 × 107 particles/mL. Overall, the multivalent binding ability of TMAP and the signal amplification characteristics of AgNCs have a certain driving role in achieving clinical detection of exosomes.

Self-Regulatory Micro- and Macroscale Patterning of ATP-Mediated Nanobioconjugate.

Directional interactions and the assembly of a nanobioconjugate in clusters at a specific location are important for patterning and microarrays in biomedical research. Herein, we report that self-assembly and spatial control in surface patterning of the surfactant-functionalized nanoparticles can be governed in micro- and macroscale environments by two factors, synergistic enzyme-substrate-nanoparticle affinity and the phoretic effect. First, we show that aggregation of cationic gold nanoparticles (GNP) can be modulated by multivalent anionic nanoparticle binding of an adenosine-based nucleotide and enzyme, alkaline phosphatase. We further demonstrate two different types of their autonomous aggregation pattern: (i) by introducing an enzyme gradient that modulates the synergistic nonequilibrium interactivity of the nanoparticle, nucleotide, and enzyme both in microfluidic conditions and at the macroscale; and (ii) the surface deposition pattern from evaporating droplets via the coffee ring effect. Here, temporal control over the width and site of the patterning area inside the microfluidic channel under catalytic and noncatalytic conditions has also been demonstrated. Finally, we show a change in capillary phoresis parameters responsible for the coffee ring due to introduction of ATP-loaded GNP in the blood serum, showing applicability in low-cost disease diagnostics. Overall, an enzyme-actuated surface nanobiopatterning method has been demonstrated that has potential application in controlled micro- and macroscale area patterning with a diverse cascade catalytic surface and spatiotemporal multisensory-based application.

Flexible Organic Photodetectors with Mechanically Robust Zinc Oxide Nanoparticle Thin Films.

Zinc oxide nanoparticle (ZnO-NP) thin films have been intensively used as electron transport layers (ETLs) in organic optoelectronic devices, but their moderate mechanical flexibility hinders their application to flexible electronic devices. This study reveals that the multivalent interaction between ZnO-NPs and multicharged conjugated electrolytes, such as diphenylfluorene pyridinium bromide derivative (DFPBr-6), can significantly improve the mechanical flexibility of ZnO-NP thin films. Intermixing ZnO-NPs and DFPBr-6 facilitates the coordination between bromide anions (from the DFPBr-6) and zinc cations on ZnO-NP surfaces, forming Zn2+-Br- bonds. Different from a conventional electrolyte (e.g., KBr), DFPBr-6 with six pyridinium ionic side chains holds the Br--chelated ZnO-NPs adjacent to DFP+ through Zn2+-Br--N+ bonds. Consequently, ZnO-NP:DFPBr-6 thin films exhibit improved mechanical flexibility with a critical bending radius as low as 1.5 mm under tensile bending conditions. Flexible organic photodetectors with ZnO-NP:DFPBr-6 thin films as ETLs demonstrate reliable device performances with high R (0.34 A/W) and D* (3.03 × 1012 Jones) even after 1000 times repetitive bending at a bending radius of 4.0 mm, whereas devices with ZnO-NP and ZnO-NP:KBr ETLs yield >85% reduction in R and D* under the same bending condition.

"Hook&Loop" multivalent interactions based on disk-shaped nanoparticles strengthen active targeting.

How to enhance active targeting efficiency remains a challenge. Multivalent interactions play a crucial role in improving the binding ability between ligands and receptors. It is hypothesized that nanoparticles bearing a flat conformation attain simultaneous formation of multiple ligand-receptor bindings, which could be vividly metaphorized by the "Hook&Loop" rationale. In this study, spherical, rod-shaped and disk-shaped folic acid-modified red blood cell membrane-coated biomimetic mesoporous silica nanoparticles (FRMSNs) were prepared to verify the shape-based multivalent interactions. The fundamental concepts of multivalent interactions have been proved by a series of both in vitro and in vivo evaluations. Physical characterization confirmed the morphology, shape and surface features of FRMSNs. Strengthened binding and internalization of disk-shaped FRMSNs by K562 cells stresses the merits of multivalent interactions. Whereas Bio-TEM visually demonstrates the proposed "plane" contact of disk-shaped particles with cells, quantification further confirmed strengthened "plane" binding affinity with folate binding proteins owing to multivalent interactions. In K562 xenograft mice, doxorubicin-loaded disk-shaped FRMSNs effectively slowed down chronic myeloid leukemia progression. It is concluded that disks favor multivalent interactions which leads to enhanced active targeting efficiency.

Recapitulating the Lateral Organization of Membrane Receptors at the Nanoscale.

Many cell membrane functions emerge from the lateral presentation of membrane receptors. The link between the nanoscale organization of the receptors and ligand binding remains, however, mostly unclear. In this work, we applied surface molecular imprinting and utilized the phase behavior of lipid bilayers to create platforms that recapitulate the lateral organization of membrane receptors at the nanoscale. We used liposomes decorated with amphiphilic boronic acids that commonly serve as synthetic saccharide receptors and generated three lateral modes of receptor presentation─random distribution, nanoclustering, and receptor crowding─and studied their interaction with saccharides. In comparison to liposomes with randomly dispersed receptors, surface-imprinted liposomes resulted in more than a 5-fold increase in avidity. Quantifying the binding affinity and cooperativity proved that the boost was mediated by the formation of the nanoclusters rather than a local increase in the receptor concentration. In contrast, receptor crowding, despite the presence of increased local receptor concentrations, prevented multivalent oligosaccharide binding due to steric effects. The findings demonstrate the significance of nanometric aspects of receptor presentation and generation of multivalent ligands including artificial lectins for the sensitive and specific detection of glycans.

Biological condensates form percolated networks with molecular motion properties distinctly different from dilute solutions.

Formation of membraneless organelles or biological condensates via phase separation and related processes hugely expands the cellular organelle repertoire. Biological condensates are dense and viscoelastic soft matters instead of canonical dilute solutions. To date, numerous different biological condensates have been discovered, but mechanistic understanding of biological condensates remains scarce. In this study, we developed an adaptive single-molecule imaging method that allows simultaneous tracking of individual molecules and their motion trajectories in both condensed and dilute phases of various biological condensates. The method enables quantitative measurements of concentrations, phase boundary, motion behavior, and speed of molecules in both condensed and dilute phases, as well as the scale and speed of molecular exchanges between the two phases. Notably, molecules in the condensed phase do not undergo uniform Brownian motion, but instead constantly switch between a (class of) confined state(s) and a random diffusion-like motion state. Transient confinement is consistent with strong interactions associated with large molecular networks (i.e., percolation) in the condensed phase. In this way, molecules in biological condensates behave distinctly different from those in dilute solutions. The methods and findings described herein should be generally applicable for deciphering the molecular mechanisms underlying the assembly, dynamics, and consequently functional implications of biological condensates.

Multivalent peptide dendrimers inhibit the fusion of viral-cellular membranes and the cellular NF-κB signaling pathway.

The binding of the influenza A virus (IAV) to host cells is multivalent interactions between the hemagglutinin (HA) trimer and sialic acid residues on the cell surface, which present a challenge for the development of efficient antiviral drugs interfering with the entry of IAV into host cells. In this study, a number of multivalent peptide dendrimers targeting the HA2 subunit of HA to block the fusion between viral-cellular membranes have been created, of which FMOC-4-KKWK showed the lowest cytotoxicity, while in the nanomolar concentration range of antiviral effects. In addition to being active against a panel of various subtypes of influenza viruses, these dendrimers reduced the levels of NF-κB in RAW 264.7 cells and inhibited the overexpression of proinflammatory cytokines of TNF-α, IL-1ß, and IL-6 that are associated with the influenza infection. Further tests in mice infected with a lethal dose of PR8 virus showed that these dendrimers increased the survival rate of mice, and reduced the viral load in the lungs. Significantly, this is the first report describing peptide dendrimers that target the HA2 subunit of IAV, differing from those using carbohydrates as ligands to block the adsorption of viruses to host cells.

Fabrication of a Polymeric Inhibitor of Proximal Metabolic Enzymes in Hypoxia for Synergistic Inhibition of Cancer Cell Proliferation, Survival, and Migration.

Since conventional molecular targeted drugs often result in side effects, the development of novel molecular targeted drugs with both high efficacy and selectivity is desired. Simultaneous inhibition of metabolically and spatiotemporally related proteins/enzymes is a promising strategy for improving therapeutic interventions in cancer treatment. Herein, we report a poly-α-l-glutamate-based polymer inhibitor that simultaneously targets proximal transmembrane enzymes under hypoxia, namely, carbonic anhydrase IX (CAIX) and zinc-dependent metalloproteinases. A polymer incorporating two types of inhibitors more effectively inhibited the proliferation and migration of human breast cancer cells than a combination of two polymers functionalized exclusively with either inhibitor. Synergistic inhibition of cancer cells would occur owing to the hetero-multivalent interactions of the polymer with proximate enzymes on the cancer cell membrane. Our results highlight the potential of polymer-based cancer therapeutics.

Development of novel 67/68Ga-labeled pyridyl benzofuran derivatives as islet amyloid imaging probes.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) accounts for the majority of diabetes mellitus cases, and ß-cell function and mass may be involved in its pathophysiology. The deposition of islet amyloid in pancreas islets is one of the mechanisms of progression of T2DM. Therefore, the development of imaging techniques for islet amyloid deposition contributes to the early-phase diagnosis and elucidation of the pathogenic mechanism of T2DM. We have reported that pyridyl benzofuran (PBF) is a promising scaffold for islet amyloid imaging probes. In this study, we designed [67/68Ga]Ga-dedpa-(PBF)2, which contains two PBF scaffolds in one molecule for enhancing binding affinity for islet amyloid by multivalent interaction, and evaluated its utility as an islet amyloid imaging probe. METHODS: We synthesized dedpa-PBF and dedpa-(PBF)2 as mono and bivalent compounds, respectively. We conducted an in vitro saturation binding assay for calculation of the dissociation constants (Kd) against islet amyloid aggregates. An in vitro autoradiographic study was performed using pancreatic tissue sections of T2DM patients. A biodistribution experiment was performed using normal mice. Finally, we carried out an ex vivo autoradiographic study using islet amyloid-transplanted model mice. RESULTS: In the in vitro saturation binding assay, the affinity of [67Ga]Ga-dedpa-(PBF)2 (Kd = 2.46 ± 0.49 µM) for islet amyloid aggregates was higher than that of [67Ga]Ga-dedpa-PBF (Kd = 3.73 ± 1.75 µM). The in vitro autoradiographic study revealed that both compounds clearly labeled islet amyloid depositions in T2DM sections. In the biodistribution experiment using normal mice, both compounds had initial uptake in the pancreas, but retention of radioactivity was observed in the pancreas and surrounding organs. In ex vivo autoradiography, both compounds exhibited intensive accumulation of radioactivity, which corresponded to Thioflavin S staining in amylin-transplanted mouse pancreas. CONCLUSION: [67/68Ga]Ga-dedpa-(PBF)2 demonstrated the fundamental characteristics of an islet amyloid imaging probe, although it is necessary to improve the clearance from organs.

Multivalent display of engineered HIV-1 envelope trimers on silica nanoparticles for targeting and in vitro activation of germline VRC01 B cells.

Selective targeting of germline B cells with specifically designed germline-targeting HIV-1 envelope immunogens (GT-Env) is considered a feasible vaccination strategy to elicit broadly neutralizing antibodies (bnAbs). BnAbs are extremely valuable because they neutralize genetically distant viral strains at the same time. To overcome its inherently low affinity to germline B cells, the aim of the study was to present GT-Env via different immobilization strategies densely arrayed on the surface of nanoparticles. We engineered a prefusion-stabilized GT-Env trimer with affinity to VRC01 germline B cells using a bioinformatics-supported design approach. Distinct glycan modifications and amino acid substitutions yielded a GT-Env trimer which bound to the receptor with a KD of 11.5 µM. Silica nanoparticles with 200 nm diameter (SiNPs) were used for the multivalent display of the novel GT-Env with a 15 nm mean centre-to-centre spacing either by site-specific, covalent conjugation or at random, non-specific adsorption. Oriented, covalent GT-Env conjugation revealed better binding of structure dependent bnAbs as compared to non-specifically adsorbed GT-Env. In addition, GT-Env covalently attached activated a B cell line expressing the germline VRC01 receptor at an EC50 value in the nanomolar range (4 nM), while soluble GT-Env required 1,000-fold higher concentrations to induce signalling. The significantly lower GT-Env concentration was likely required due to avidity effects, which were in the picomolar range. Thus, low affinity antigens may particularly benefit from a particulate and multivalent delivery. In future, SiNPs are ideal to be modified in a modular design with various GT-Env variants that target different stages of germline and bnAb precursor B cells.

Acid-Sensitive Supramolecular Nanoassemblies with Multivalent Interaction: Effective Tumor Retention and Deep Intratumor Infiltration.

It remains a conundrum to reconcile the contradiction between effective tumor retention and deep intratumor infiltration for nanotherapeutics due to the sophisticated drug delivery journey. Herein, we reported an acid-sensitive supramolecular nanoassemblies (DCD SNs) based on the multivalent host-gest inclusions of two polymer conjugates for conquering diverse physiological blockages and amplifying therapeutic efficacy. The multiple inclusions of repetitive units on the hydrophilic polymer backbone reinforced the binding affinity and induced robust self-assembly, ameliorating instability of the self-assemblies and facilitating to prolong the drug retention time. By virtue of the acid-sensitive Schiff base linkages, the supramolecular nanoassembly could respond to the unique tumor microenvironment (TME), dissociate, and transform into smaller particles (∼30 nm), thereby efficiently traversing the complicated extracellular matrix and irregular blood vessels to achieve deep intratumor infiltration. The acid-sensitive DCD SNs can absorb a large number of protons in the acidic lysosomal environment, causing the proton sponge effect, which was conducive to their escape from endolysosomes and accelerated lysosomal disruption, so that the active chemotherapeutic doxorubicin (DOX) could enter the nucleus well and exert severe DNA damage to induce apoptosis. This versatile supramolecular nanoplatform is anticipated to be a promising candidate to overcome the limitations of insufficient stability within the circulation and weak intratumor penetration.