Spatiotemporal Organization of Functional Cargoes by Light-Switchable Condensation in Escherichia coli Cells.

Biomolecular condensates are dynamic subcellular compartments that lack surrounding membranes and can spatiotemporally organize the cellular biochemistry of eukaryotic cells. However, such dynamic organization has not been realized in prokaryotes that naturally lack organelles, and strategies are urgently needed for dynamic biomolecular compartmentalization. Here we develop a light-switchable condensate system for on-demand dynamic organization of functional cargoes in the model prokaryotic Escherichia coli cells. The condensate system consists of two modularly designed and genetically encoded fusions that contain a condensation-enabling scaffold and a functional cargo fused to the blue light-responsive heterodimerization pair, iLID and SspB, respectively. By appropriately controlling the biogenesis of the protein fusions, the condensate system allows rapid recruitment and release of cargo proteins within seconds in response to light, and this process is also reversible and repeatable. Finally, the system is demonstrated to dynamically control the subcellular localization of a cell division inhibitor, SulA, which enables the reversible regulation of cell morphologies. Therefore, this study provides a new strategy to dynamically control cellular processes by harnessing light-controlled condensates in prokaryotic cells.

Active secretion of IL-33 from astrocytes is dependent on TMED10 and promotes central nervous system homeostasis.

Interleukin-33 (IL-33), secreted by astrocytes, regulates the synapse development in the spinal cord and hippocampus and suppresses autoimmune disease in the central nervous system (CNS). However, the mechanism of unconventional protein secretion of this cytokine remains unclear. In this study, we found that IFN-γ promotes the active secretion of IL-33 from astrocytes, and the active secretion of IL-33 from cytoplasm to extracellular space was dependent on interaction with transmembrane emp24 domain 10 (TMED10) via the IL-1 like cytokine domain in astrocytes. Knockout of Il-33 or its receptor St2 induced hippocampal astrocyte activation and depressive-like disorder in naive mice, as well as increased spinal cord astrocyte activation and polarization to a neurotoxic reactive subtype and aggravated passive experimental autoimmune encephalomyelitis (EAE). Our results have identified that IL-33 is actively secreted by astrocytes through the unconventional protein secretion pathway facilitated by TMED10 channels. This process helps maintain CNS homeostasis by inhibiting astrocyte activation.

Modulating Polyalanine Motifs of Synthetic Spidroin for Controllable Preassembly and Strong Fiber Formation.

Spider dragline (major ampullate) silk is one of the toughest known fibers in nature and exhibits an excellent combination of high tensile strength and elasticity. Increasing evidence has indicated that preassembly plays a crucial role in facilitating the proper assembly of silk fibers by bridging the mesoscale gap between spidroin molecules and the final strong fibers. However, it remains challenging to control the preassembly of spidroins and investigate its influence on fiber structural and mechanical properties. In this study, we explored to bridge this gap by modulating the polyalanine (polyA) motifs in repetitive region of spidroins to tune their preassemblies in aqueous dope solutions. Three biomimetic silk proteins with varying numbers of alanine residues in polyA motif and comparable molecular weights were designed and biosynthesized, termed as N16C-5A, N15C-8A, and N13C-12A, respectively. It was found that all three proteins could form nanofibril assemblies in the concentrated aqueous dopes, but the size and structural stability of the fibrils were distinct from each other. The silk protein N15C-8A with 8 alanine residues in polyA motif allowed for the formation of stable nanofibril assemblies with a length of approximately 200 nm, which were not prone to disassemble or aggregate as that of N16C-5A and N13C-12A. More interestingly, the stable fibril assembly of N15C-8A enabled spinning of simultaneously strong (623.3 MPa) and tough (107.1 MJ m-3) synthetic fibers with fine molecular orientation and close interface packing of fibril bundles. This work highlights that modulation of polyA motifs is a feasible way to tune the morphology and stability of the spidroin preassemblies in dope solutions, thus controlling the structural and mechanical properties of the resulting fibers.

Designer protein compartments for microbial metabolic engineering.

Protein compartments are distinct structures assembled in living cells via self-assembly or phase separation of specific proteins. Significant efforts have been made to discover their molecular structures and formation mechanisms, as well as their fundamental roles in spatiotemporal control of cellular metabolism. Here, we review the design and construction of synthetic protein compartments for spatial organization of target metabolic pathways toward increased efficiency and specificity. In particular, we highlight the compartmentalization strategies and recent examples to speed up desirable metabolic reactions, to reduce the accumulation of toxic metabolic intermediates, and to switch competing metabolic pathways. We also identify the most important challenges that need to be addressed for exploitation of these designer compartments as a versatile toolkit in metabolic reprogramming.

Programmable adhesion and morphing of protein hydrogels for underwater robots.

Soft robots capable of efficiently implementing tasks in fluid-immersed environments hold great promise for diverse applications. However, it remains challenging to achieve robotization that relies on dynamic underwater adhesion and morphing capability. Here we propose the construction of such robots with designer protein materials. Firstly, a resilin-like protein is complexed with polyoxometalate anions to form hydrogels that can rapidly switch between soft adhesive and stiff non-adhesive states in aqueous environments in response to small temperature variation. To realize remote control over dynamic adhesion and morphing, Fe3O4 nanoparticles are then integrated into the hydrogels to form soft robots with photothermal and magnetic responsiveness. These robots are demonstrated to undertake complex tasks including repairing artificial blood vessel, capturing and delivering multiple cargoes in water under cooperative control of infrared light and magnetic field. These findings pave an avenue for the creation of protein-based underwater robots with on-demand functionalities.

IL-33 and HMGB1 modulate the progression of EAE via oppositely regulating each other.

Interleukin-33 (IL-33) and high mobility group box 1 (HMGB1) have been reported to play crucial and distinct roles in experimental autoimmune encephalomyelitis (EAE). However, little is known about their interaction in the progression of EAE. In this study, the dynamic expression and release of IL-33 and HMGB1 in different stages of EAE in vivo, and their interaction in vitro were explored. We found that HMGB1 was dominant in pre-onset stage of EAE, while IL-33 was dominant in peak stage. Moreover, both blockade of extracellular HMGB1 in the central nervous system (CNS) and conditional knockout of HMGB1 in astrocytes decreased IL-33 release. HMGB1 promoted the release of IL-33, while IL-33 reduced the release of HMGB1 from primary astrocytes in vitro. Taken together, IL-33 and HMGB1 in the CNS jointly participate in the EAE progression and the inhibitory effect of IL-33 on HMGB1 may be involved in the self-limiting of EAE.

Amphiphilicity-Driven Small Alcohols Regulate the Flexibility of Pesticide-Loaded Microcapsules for Better Foliar Adhesion and Utilization.

The foliar loss of pesticides causes serious utilization decline and environmental risk. On the basis of biomimetics, pesticide-loaded microcapsules (MCs) with spontaneous deformation on foliar micro/nanostructures, like the snail suction cup, are prepared by interfacial polymerization. By controlling the usage or types of small alcohols in the MC preparation system, the flexibility of MCs is tunable. Through the investigation of emulsions and MC structures, we discover that the migration and distribution of small alcohols driven by amphiphilicity affect the process of interfacial polymerization between polyethylene glycol and 4,4-methylenediphenyl diisocyanate. By hydrophobic modification of the polymer and competition for oil monomers of small alcohols, the thickness and compactness of shells are reduced, whereas the density of the core is increased. As a result of the regulation in structures, the flexibility of MCs is improved significantly. In particularly, the MCs-N-pentanol (0.1 mol kg-1) with the best flexibility show strong scouring resistance on varied foliar structures, sustained release property on the air/solid interface, and persistent control effect against foliar diseases. The pesticide-loaded soft MCs provide an effective way to improve pesticide foliar utilization.

IL-33 Contributes to the Pathological Changes of Hair Follicles in Psoriasis: A Potential Target for Psoriatic Alopecia.

Purpose: IL-33 is constitutively expressed in skin tissues. Alopecia, a T cells-driven disorder of the hair follicles (HFs), is a common complication in the development of psoriasis. However, the role of IL-33 in psoriatic alopecia remains uncovered. Here, we investigated the roles of IL-33 in inducing pathological changes of hair follicles in psoriasis. Patients and Methods: Clinical samples and imiquimod (IMQ)-induced psoriatic mice samples were used to investigate the pathological changes and T-cell infiltration of HFs. By using immunohistochemistry staining, the distribution and expression alteration of IL-33 in HFs were determined. Next, by using IL-33 and ST2 knockout mice, we investigated the role of IL-33/ST2 axis in the pathological changes of HFs in psoriasis. Meanwhile, recombinant IL-33 protein was subcutaneous injected to confirm its effect. Finally, RNA sequencing was used to clarify the genes and signaling pathways that involved in this process. Differentially expressed genes were further verified by RT-PCR in cultured HFs in vitro. Results: We found that the pathological changes of HFs and T cells infiltration in imiquimod-induced psoriatic mice were similar to that in psoriasis patients. The IL-33 positive keratinocytes in the outer root sheath of HFs were increased in both psoriasis patients and psoriatic model mice compared with the controls. By using gene knockout mice, we found that the pathological changes and T cell infiltration were attenuated in IL-33-/- and ST2-/- psoriatic model mice. In addition, subcutaneous injection of recombinant IL-33 exacerbated the pathological changes of HFs and T cell infiltration. RNA sequencing and RT-RCR revealed that IL-33 upregulated the transcription of genes related to keratinocytes proliferation and T lymphocytes chemotaxis. Conclusion: Our study identifies that IL-33 promotes the pathological changes of HFs in psoriasis, which contributes to psoriatic alopecia. Inhibition of IL-33 may be a potential therapeutic approach for psoriatic alopecia.

Direct and Efficient Incorporation of DOPA into Resilin-Like Proteins Enables Cross-Linking into Tunable Hydrogels.

3,4-Dihydroxyphenylalanine (DOPA), a naturally occurring yet noncanonical amino acid, endows protein polymers with diverse chemical reactivities and novel functionalities. Although many efforts have been made to incorporate DOPA into proteins, the incorporation efficiency and production titer remain low and severely hinder the exploration of these peculiar proteins for biomaterial fabrication. Here, we report an efficient biosynthetic strategy to produce large amounts of DOPA-incorporated structural proteins for the fabrication of hydrogels with tunable mechanical properties. First, synthetic genes were constructed that encode repetitive resilin-like proteins (RLPs) with varying proportions of tyrosine residues and molecular weights (Mw). Decoding of these genes into RLPs incorporated with DOPA was achieved via mis-aminoacylation of DOPA by endogenous tyrosyl-tRNA synthetase (TyrRS) in recombinant Escherichia coli cells. By developing a stoichiometry-guided two-phase culture strategy, we achieved independent control of the bacterial growth and protein synthesis phases. This enabled hyperproduction of the DOPA-incorporated RLPs at gram-per-liter levels and with a high DOPA incorporation yield of 76-85%. The purified DOPA-containing RLPs were then successfully cross-linked into bulk hydrogels via facile DOPA-Fe3+ complexations. Interestingly, these hydrogels exhibited viscoelastic and self-healing properties that are highly dependent on the catechol content and Mw of the RLPs. Finally, exploration of the molecular cross-linking mechanisms revealed that higher DOPA contents of the proteins would result in the concomitant occurrence of metal coordination and oxidative covalent cross-linking. In summary, our results suggest a useful platform to generate DOPA-functionalized protein materials and provide deeper insights into the gelation systems based on DOPA chemistry.

Characterization of Dextran Biosynthesized by Glucansucrase from Leuconostoc pseudomesenteroides and Their Potential Biotechnological Applications.

Glucansucrase was purified from Leuconostoc pseudomesenteroides. The glucansucrase exhibited maximum activity at pH 5.5 and 30 °C. Ca2+ significantly promoted enzyme activity. An exopolysaccharide (EPS) was synthesized by this glucansucrase in vitro and purified. The molecular weight of the EPS was 3.083 × 106 Da. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy showed that the main structure of glucan was 97.3% α-(1→6)-linked D-glucopyranose units, and α-(1→3) branched chain accounted for 2.7%. Scanning electron microscopy (SEM) observation of dextran showed that its surface was smooth and flaky. Atomic force microscopy (AFM) of dextran revealed a chain-like microstructure with many irregular protuberances in aqueous solution. The results showed that dextran had good thermal stability, water holding capacity, water solubility and emulsifying ability (EA), as well as good antioxidant activity; thus it has broad prospects for development in the fields of food, biomedicine, and medicine.

Synthetic biology-guided design and biosynthesis of protein polymers for delivery.

Vehicles derived from genetically engineered protein polymers have gained momentum in the field of biomedical engineering due to their unique designability, remarkable biocompatibility and excellent biodegradability. However, the design and production of these protein polymers with on-demand sequences and supramolecular architectures remain underexplored, particularly from a synthetic biology perspective. In this review, we summarize the state-of-the art strategies for constructing the highly repetitive genes encoding the protein polymers, and highlight the advanced approaches for metabolically engineering expression hosts towards high-level biosynthesis of the target protein polymers. Finally, we showcase the typical protein polymers utilized to fabricate delivery vehicles.

Synthetic protein condensates for cellular and metabolic engineering.

Protein condensates are distinct structures assembled in living cells that concentrate molecules via phase separation in a confined subcellular compartment. In the past decade, remarkable advances have been made to discover the fundamental roles of the condensates in spatiotemporal control of cellular metabolism and physiology and to reveal the molecular principles, components and driving forces that underlie their formation. Here we review the unique properties of the condensates, the promise and hurdles for harnessing them toward purposeful design and manipulation of biological functions in living cells. In particular, we highlight recent advances in mining and understanding the proteinaceous components for creating designer condensates, along with the engineering approaches to manipulate their material properties and biological functions. With these advances, a greater variety of complex organelle-like structures can be built for diverse applications, with unprecedented effects on synthetic biology.

Spatially Directed Biosynthesis of Quantum Dots via Spidroin Templating in Escherichia coli.

Spatially directed synthesis of quantum dots (QDs) is intriguing yet challenging in organisms, due to the dispersed feature of templating biomolecules and precursors. Whether this task could be accomplished by biomolecular condensates, an emerging type of membraneless compartments in cells remains unknown. Here we report synthetic protein condensates for templated synthesis of QDs in bacterium Escherichia coli. This was realized by overexpression of spider silk protein to bind precursor ions and recruit other necessary components, which induced the spidroin to form more ß-sheet structures for assembly and maturation of the protein condensates. This in turn enabled formation and co-localization of the fluorescent QDs to "light up" the condensates, and alleviated cytotoxicity of the precursor heavy metal ions and resulting QDs. Thus, our results suggest a new strategy for nanostructure synthesis and deposition in subcellular compartments with great potential for in situ applications.

Dual role of injectable curcumin-loaded microgels for efficient repair of osteoarthritic cartilage injury.

Curcumin has been widely used for the treatment of age-associated diseases, and showed chondroprotective potential for post-traumatic osteoarthritis (OA). However, due to the irregular-shaped and large-sized defects on joint cartilage in degenerated OA, the in vivo delivery and therapeutic effect of curcumin for effective repair remain challenging. In this study, we first present a PEG-GelMA [Poly(Ethylene Glycol) Dimethacrylate-Gelatin Methacrylate, PGMs] hydrogel microgel-based curcumin delivery system for both improved anti-inflammatory and pro-regenerative effects in treatment for cartilage defects. The curcumin-loaded PGMs were produced by a microfluidic system based on light-induced gelation of gelatin methacrylate (GelMA). This PGMs embedding curcumin at a relative low dosage were demonstrated to promote the proliferation and chondrogenic differentiation of mesenchymal stem cells in vitro. More importantly, the PGMs were shown to attenuate the inflammatory response of chondrocytes under IL-1ß stimulation. Lastly, the in vivo application of the injectable PGMs significantly promoted the repair of large-sized cartilage injury. These results confirmed that curcumin-loaded PGMs can not only enhance the chondroprotective efficacy under inflammatory conditions but also induce efficient cartilage regeneration. This study provides an advanced strategy with anti-inflammatory and pro-regenerative dual-role therapeutic for treatment of extensive cartilage injuries.

Functionalization and Reinforcement of Recombinant Spider Dragline Silk Fibers by Confined Nanoparticle Formation.

Spider dragline silk is a remarkable protein fiber that is mechanically superior to almost any other natural or synthetic material. As a sustainable supply of natural dragline silk is not feasible, recombinant production of silk fibers with native-like mechanical properties and non-native physiochemical functions is highly desirable for various applications. Here, we report a new strategy for simultaneous functionalization and reinforcement of recombinant spider silk fibers by confined nanoparticle formation. First, a mimic silk protein (N16C) of spider Trichonephila clavipes was recombinantly produced and wet-spun into fibers. Drawing the as-spun fibers in water led to post-drawn fibers more suitable for the templated synthesis of nanoparticles (NPs) with uniform distribution throughout the synthetic fibers. This was exemplified using a chemical precipitation reaction to generate copper sulfide nanoparticle-incorporated fibers. These fibers and the derived fabric displayed a significant photothermal effect as their temperatures could increase to over 40 °C from room temperature within 3 min under near-infrared laser irradiation or simulated sunlight. In addition, the tensile strength and toughness of the nanofunctionalized fibers were greatly enhanced, and the toughness of these synthetic fibers could reach 160.1 ± 21.4 MJ m-3, which even exceeds that of natural spider dragline silk (111.19 ± 30.54 MJ m-3). Furthermore, the confined synthesis of gold NPs via a redox reaction was shown to improve the ultraviolet-protective effect and tensile mechanical properties of synthetic silk fibers. These results suggest that our strategy may have great potential for creating functional and high-performance spider silk fibers and fabrics for wide applications.

Fibrous Structure and Stiffness of Designer Protein Hydrogels Synergize to Regulate Endothelial Differentiation of Bone Marrow Mesenchymal Stem Cells.

Matrix stiffness and fibrous structure provided by the native extracellular matrix have been increasingly appreciated as important cues in regulating cell behaviors. Recapitulating these physical cues for cell fate regulation remains a challenge due to the inherent difficulties in making mimetic hydrogels with well-defined compositions, tunable stiffness, and structures. Here, we present two series of fibrous and porous hydrogels with tunable stiffness based on genetically engineered resilin-silk-like and resilin-like protein polymers. Using these hydrogels as substrates, the mechanoresponses of bone marrow mesenchymal stem cells to stiffness and fibrous structure were systematically studied. For both hydrogel series, increasing compression modulus from 8.5 to 14.5 and 23 kPa consistently promoted cell proliferation and differentiation. Nonetheless, the promoting effects were more pronounced on the fibrous gels than their porous counterparts at all three stiffness levels. More interestingly, even the softest fibrous gel (8.5 kPa) allowed the stem cells to exhibit higher endothelial differentiation capability than the toughest porous gel (23 kPa). The predominant role of fibrous structure on the synergistic regulation of endothelial differentiation was further explored. It was found that the stiffness signal activated Yes-associated protein (YAP), the main regulator of endothelial differentiation, via spreading of focal adhesions, whereas fibrous structure reinforced YAP activation by promoting the maturation of focal adhesions and associated F-actin alignment. Therefore, our results shed light on the interplay of physical cues in regulating stem cells and may guide the fabrication of designer proteinaceous matrices toward regenerative medicine.

Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers.

Spider dragline silk is a remarkable fiber made of unique proteins-spidroins-secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (>65.8%) and titer (554.7 mg L-1) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m-3. The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials.

Fraxin Prevents Knee Osteoarthritis through Inhibiting Chondrocyte Apoptosis in an Experimental Rat Osteoarthritis Model.

BACKGROUND: Current treatments of osteoarthritis are unsatisfied, a new approach towards the treatment of osteoarthritis is urged considering the state at present. OBJECTIVE: The objective of this study is to investigate the effect of fraxin on knee OA in a rat model and probe into the possible molecular mechanism. METHODS: Primary Murine Chondrocytes were isolated and cell apoptosis analyses were performed. Rat OA models were established using meniscectomy method and allocated into three groups. Knee joint specimens were collected for qRT-PCR, western blotting and histological analysis. Statistical analyses were processed by using a SPSS. RESULTS: The apoptosis rate of fraxin group is significantly reduced compared with the OA group or the control group. Fraxin remarkably down-regulated the expression of cleaved-Caspase-3 while significantly up-regulated the expression of Bcl-2, both on mRNA and protein levels. Toluidine blue stain results show relatively lighter articular cartilage damage compared with OA group. CONCLUSION: Fraxin prevents knee osteoarthritis by inhibiting chondrocyte apoptosis, which makes it a potential candidate as an anti-OA drug for clinical use.

3D electron-beam writing at sub-15 nm resolution using spider silk as a resist.

Electron beam lithography (EBL) is renowned to provide fabrication resolution in the deep nanometer scale. One major limitation of current EBL techniques is their incapability of arbitrary 3d nanofabrication. Resolution, structure integrity and functionalization are among the most important factors. Here we report all-aqueous-based, high-fidelity manufacturing of functional, arbitrary 3d nanostructures at a resolution of sub-15 nm using our developed voltage-regulated 3d EBL. Creating arbitrary 3d structures of high resolution and high strength at nanoscale is enabled by genetically engineering recombinant spider silk proteins as the resist. The ability to quantitatively define structural transitions with energetic electrons at different depths within the 3d protein matrix enables polymorphic spider silk proteins to be shaped approaching the molecular level. Furthermore, genetic or mesoscopic modification of spider silk proteins provides the opportunity to embed and stabilize physiochemical and/or biological functions within as-fabricated 3d nanostructures. Our approach empowers the rapid and flexible fabrication of heterogeneously functionalized and hierarchically structured 3d nanocomponents and nanodevices, offering opportunities in biomimetics, therapeutic devices and nanoscale robotics.

Unconventional Spidroin Assemblies in Aqueous Dope for Spinning into Tough Synthetic Fibers.

Spider dragline silk is a remarkable fiber made by spiders from an aqueous solution of spidroins, and this feat is largely attributed to the tripartite domain architecture of the silk proteins leading to the hierarchical assembly at the nano- and microscales. Although individual amino- and carboxy-terminal domains have been proposed to relate to silk protein assembly, their tentative synergizing roles in recombinant spidroin storage and spinning into synthetic fibers remain elusive. Here, we show biosynthesis and self-assembly of a mimic spidroin composed of amino- and carboxy-terminal domains bracketing 16 consensus repeats of the core region from spider Trichonephila clavipes. The presence of both termini was found essential for self-assembly of the mimic spidroin termed N16C into fibril-like (rather than canonical micellar) nanostructures in concentrated aqueous dope and ordered alignment of these nanofibrils upon extrusion into an acidic coagulation bath. This ultimately led to continuous, macroscopic fibers with a tensile fracture toughness of 100.9 ± 13.2 MJ m-3, which is comparable to that of their natural counterparts. We also found that the recombinant proteins lacking one or both termini were unable to similarly preassemble into fibrillar nanostructures in dopes and thus yielded inferior fiber properties. This work thereby highlights the synergizing role of terminal domains in the storage and processing of recombinant analogues into tough synthetic fibers.