Miao Liu

Bio: Miao Liu is an academic researcher from The Chinese University of Hong Kong. The author has contributed to research in topics: PDZ domain & Peptide. The author has an hindex of 5, co-authored 7 publications receiving 72 citations.

Self-Assembled Multienzyme Nanostructures on Synthetic Protein Scaffolds

Abstract: Sequential enzymes in a biosynthetic pathway often self-assemble to form nanomachineries known as multienzyme complexes inside cells. Enzyme self-assembly insulates toxic intermediates, increases the efficiency of intermediate transfer, minimizes metabolic crosstalk, streamlines flux, and improves the product yield. Artful structures and superior catalytic functions of these natural nanomachines inspired the development of synthetic multienzyme complexes to expedite biosynthesis. Here we present a versatile self-assembly strategy to construct multienzyme nanostructures based on synthetic protein scaffolds. The protein scaffolds were formed using the spontaneous protein reaction of SpyCatcher and SpyTag. Two types of protein scaffolds were generated: two skeleton proteins cross-linked and hierarchically assembled into heterogeneous nanostructures (the cross-linked scaffold), and head-to-tail cyclization of a dual-reactive skeleton protein gave a homogeneous cyclic scaffold. Sequential enzymes from the menaquinone biosynthetic pathway were assembled on both scaffolds through the docking domain interactions derived from polyketide synthases. Both scaffolded assemblies effectively increased the yield of the final product of the cascade catalytic reaction in menaquinone biosynthesis. Surprisingly, the rate enhancements were driven by different mechanisms: the cross-linked scaffold assembly streamlined the overall flow of the reactants, whereas the cyclic scaffold assembly accelerated the catalytic efficiency of the rate-limiting enzyme. Altogether, self-assembly of sequential enzymes by combining the SpyCatcher/SpyTag reaction and the docking domain interactions yielded protein-based nanostructures with special architecture, exceptional catalytic activity, and unexpected catalytic mechanisms. This work demonstrates a versatile strategy of gaining more powerful biocatalysts by protein self-assembly for efficient bioconversion of valuable chemicals.

PDZ-Reactive Peptide Activates Ephrin-B Reverse Signaling and Inhibits Neuronal Chemotaxis.

Abstract: Intracellular reactions on nonenzymatic proteins that activate cellular signals are rarely found. We report one example here that a designed peptide derivative undergoes a nucleophilic reaction specifically with a cytosolic PDZ protein inside cells. This reaction led to the activation of ephrin-B reverse signaling, which subsequently inhibited SDF-1 induced neuronal chemotaxis of human neuroblastoma cells and mouse cerebellar granule neurons. Our work provides direct evidence that PDZ-RGS3 bridges ephrin-B reverse signaling and SDF-1 induced G protein signaling for the first time.

Membrane-Permeable Antibacterial Enzyme against Multidrug-Resistant Acinetobacter baumannii.

Abstract: Bacteriophage endolysins (lysins, or murein hydrolases) are enzymes that bacteriophages utilize to degrade the cell wall peptidoglycans (PG) and subsequently disintegrate bacterial cells from within. Due to their muralytic activity, lysins are considered as potential candidates to battle against antibiotic resistance. However, most lysins in their native form lack the capability of trespassing the outer membrane (OM) of Gram-negative (G-ve) bacteria. To turn the bacteriophage enzymes into antibacterial weapons against G-ve bacteria, endowing these enzymes the capability of accessing the PG substrate underneath the OM is critical. Here we show that fusing a membrane-permeabilizing peptide CeA at the C-terminus of a muralytic enzyme LysAB2 renders a two-step mechanism of bacterial killing and increases the activity of LysAB2 against the multidrug resistant Acinetobacter baumannii by up to 100 000-folds. The engineered LysAB2, termed LysAB2-KWK here, also shows remarkable activity against A. baumannii at the stationary phase and a prominent capability to disrupt biofilm formation. In addition, the enzyme shows a broad antibacterial spectrum against G-ve bacteria, a decent tolerance to serum, and a prolonged storage life. LysAB2-KWK rescues the larva of the greater wax moth Galleria mellonella from A. baumannii infection through systemic administration. Altogether, our work equips a globular lysin with OM permeabilization activity to enable effective killing of G-ve bacteria, reveals the critical role of the C-terminus of a globular lysin in the antibacterial activity, and points toward a viable route to engineer globular lysins as antibacterial enzymes for potential clinical use against multidrug resistant G-ve bacteria.

Affinity Enhancement by Ligand Clustering Effect Inspired by Peptide Dendrimers−Shank PDZ Proteins Interactions

Abstract: High-affinity binders are desirable tools to probe the function that specific protein−protein interactions play in cell. In the process of seeking a general strategy to design high-affinity binders, we found a clue from the βPIX (p21-activated kinase interacting exchange factor)−Shank PDZ interaction in synaptic assembly: three PDZ-binding sites are clustered by a parallel coiled-coil trimer but bind to Shank PDZ protein with 1:1 stoichiometry (1 trimer/1 PDZ). Inspired by this architecture, we proposed that peptide dendrimer, mimicking the ligand clustering in βPIX, will also show enhanced binding affinity, yet with 1:1 stoichiometry. This postulation has been proven here, as we synthesized a set of monomeric, dimeric and trimeric peptides and measured their binding affinity and stoichiometry with Shank PDZ domains by isothermal titration calorimetry, native mass spectrometry and surface plasmon resonance. This affinity enhancement, best explained by proximity effect, will be useful to guide the design of high-affinity blockers for protein−protein interactions.

Biocatalytic cascades operating on macromolecular scaffolds and in confined environments

Abstract: Biocatalytic cascades guide complex, efficient and selective intracellular transformations. These unique features originate from the spatial organization of the biocatalysts in confined cellular environments that allow the directional channelling of reaction intermediates across the cells. Here we address efforts directed towards the development of synthetic cell analogues and supramolecular ensembles acting as nano/microenvironments for operating biocatalytic cascades. Multienzyme systems are integrated within metal–organic frameworks, polymersomes, lipid-stabilized microdroplets and hydrogel microparticles acting as cell-like containments. Also, multienzyme systems are spatially positioned on one-dimensional DNA wires, two-dimensional DNA strips or origami tiles, and three-dimensional DNA origami bundles or cages, and specific protein–protein interactions or peptide–protein complexes provide versatile scaffolds for engineering enzyme assemblies. Biocatalytic cascades operating on these scaffolds or in confined nano/microenvironments reveal substantially enhanced reaction yields compared with the analogous diffusional mixtures of the biocomponents. Mechanistic pathways accounting for the enhanced biocatalytic activities and future challenges in developing and applying biocatalytic cascades are presented. Spatial organization of biocatalytic cascades can improve their performance. In this Review Article, Itamar Willner and colleagues discuss technologies to artificially confine and localize enzyme cascades, the origin of observed rate enhancements and potential applications of such designed systems.

π-Clamp-mediated cysteine conjugation

Abstract: Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the 'π-clamp', that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

Power to the protein: enhancing and combining activities using the Spy toolbox.

Abstract: Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein analysis or application. Here we review SpyTag technology, now used in hundreds of publications or patents, and its potential for detecting and controlling protein behaviour. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded. New variants of this pair allow reaction at a rate approaching the diffusion limit, while reversible versions allow purification of SpyTagged proteins or tuned dynamic interaction inside cells. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphene and the air/water interface. SpyTag/SpyCatcher is mechanically stable, so is widely used for investigating protein folding and force sensitivity. A toolbox of scaffolds allows SpyTag-fusions to be assembled into defined multimers, from dimers to 180-mers, or unlimited 1D, 2D or 3D networks. Icosahedral multimers are being evaluated for vaccination against malaria, HIV and cancer. For enzymes, Spy technology has increased resilience, promoted substrate channelling, and assembled hydrogels for continuous flow biocatalysis. Combinatorial increase in functionality has been achieved through modular derivatisation of antibodies, light-emitting diodes or viral vectors. In living cells, SpyTag allowed imaging of protein trafficking, retargeting of CAR-T cell killing, investigation of heart contraction, and control of nucleosome position. The simple genetic encoding and rapid irreversible reaction provide diverse opportunities to enhance protein function. We describe limitations as well as future directions.

Molecular Affinity Agents for Intrinsic Surface-Enhanced Raman Scattering (SERS) Sensors.

Abstract: Research at the interface of synthetic materials, biochemistry, and analytical techniques has enabled sensing platforms for applications across many research communities. Herein we review the materials used as affinity agents to create surface-enhanced Raman spectroscopy (SERS) sensors. Our scope includes those affinity agents (antibody, aptamer, small molecule, and polymer) that facilitate the intrinsic detection of targets relevant to biology, medicine, national security, environmental protection, and food safety. We begin with an overview of the analytical technique (SERS) and considerations for its application as a sensor. We subsequently describe four classes of affinity agents, giving a brief overview on affinity, production, attachment chemistry, and first uses with SERS. Additionally, we review the SERS features of the affinity agents, and the analytes detected by intrinsic SERS with that affinity agent class. We conclude with remarks on affinity agent selection for intrinsic SERS sensing platforms.

PDZ Domains as Drug Targets.

Abstract: Protein-protein interactions within protein networks shape the human interactome, which often is promoted by specialized protein interaction modules, such as the postsynaptic density-95 (PSD-95), discs-large, zona occludens 1 (ZO-1) (PDZ) domains. PDZ domains play a role in several cellular functions, from cell-cell communication and polarization, to regulation of protein transport and protein metabolism. PDZ domain proteins are also crucial in the formation and stability of protein complexes, establishing an important bridge between extracellular stimuli detected by transmembrane receptors and intracellular responses. PDZ domains have been suggested as promising drug targets in several diseases, ranging from neurological and oncological disorders to viral infections. In this review, the authors describe structural and genetic aspects of PDZ-containing proteins and discuss the current status of the development of small-molecule and peptide modulators of PDZ domains. An overview of potential new therapeutic interventions in PDZ-mediated protein networks is also provided.