Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain.

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.

A Self-Assembled Matrix System for Cell-Bioengineering Applications in Different Dimensions, Scales, and Geometries.

Stem cell bioengineering and therapy require different model systems and materials in different stages of development. If a chemically defined biomatrix system can fulfill most tasks, it can minimize the discrepancy among various setups. By screening biomaterials synthesized through a coacervation-mediated self-assembling mechanism, a biomatrix system optimal for 2D human mesenchymal stromal cell (hMSC) culture and osteogenesis is identified. Its utility for hMSC bioengineering is further demonstrated in coating porous bioactive glass scaffolds and nanoparticle synthesis for esiRNA delivery to knock down the SOX-9 gene with high delivery efficiency. The self-assembled injectable system is further utilized for 3D cell culture, segregated co-culture of hMSC with human umbilical vein endothelial cells (HUVEC) as an angiogenesis model, and 3D bioprinting. Most interestingly, the coating of bioactive glass with the self-assembled biomatrix not only supports the proliferation and osteogenesis of hMSC in the 3D scaffold but also induces the amorphous bioactive glass (BG) scaffold surface to form new apatite crystals resembling bone-shaped plate structures. Thus, the self-assembled biomatrix system can be utilized in various dimensions, scales, and geometries for many different bioengineering applications.

Noncovalent hydrogel beads as microcarriers for cell culture.

Hydrogel beads as microcarriers could have many applications in biotechnology. However, bead formation by noncovalent cross-linking to achieve high cell compatibility by avoiding chemical reactions remains challenging because of rapid gelation rates and/or low stability. Here we report the preparation of homogeneous, tunable, and robust hydrogel beads from peptide-polyethylene glycol conjugates and oligosaccharides under mild, cell-compatible conditions using a noncovalent crosslinking mechanism. Large proteins can be released from beads easily. Further noncovalent modification allows for bead labeling and functionalization with various compounds. High survival rates of embedded cells were achieved under standard cell culture conditions and after freezing the beads, demonstrating its suitability for encapsulating and conserving cells. Hydrogel beads as functional system have been realized by generating protein-producing microcarriers with embedded eGFP-secreting insect cells.

A repertoire of peptide tags for controlled drug release from injectable noncovalent hydrogel.

A repertoire of conjugable tags for controlling the release of drugs from biomaterials is highly interesting for the development of combinatorial drug administration techniques. This paper describes such a system of 11 peptide tags derived from our previous work on a physical hydrogel system cross-linked through peptide-heparin interactions. The release kinetics of the tags correlate well with their affinity to heparin and obey Fick's second law of diffusion, with the exception of the ATIII peptide, which displays a stable release profile close to a zero-order reaction. A system for release experiments over seven months was built, using the hydrogel matrix as a barrier between the reservoirs of tagged compounds and supernatant. The gel matrix can be injected without affecting the releasing properties. A tagged cyclosporin A derivative was also tested, and its release was monitored by measuring its biological activity. This work represents a design of biomaterials with an integral system of drug delivery, where both the assembly process of the matrix and affinity capture/release of tagged compounds are based on the noncovalent interaction of heparin with one class of peptides.

Minimal peptide motif for non-covalent peptide-heparin hydrogels.

Reduction of complexity of the extracellular matrix (ECM) to a non-covalent structure with minimal chemically defined components represents an attractive avenue for understanding the biology of the ECM. The resulting system could lead to the design of tailor-made biomaterials that incorporate varying functionalities. Negatively charged glycosaminoglycans are the major components of the ECM. Their interaction with positively charged proteins is important for dynamic three-dimensional scaffold formation and function. We designed and screened minimal peptide motifs whose conjugates with polyethylene glycol interact with heparin to form non-covalent hydrogels. Here we show the structure/function relationship of the (RA)(n) and (KA)(n) motifs and determined that both basic residues and the heparin-induced α-helix formation are important for the assembly process. Simple rules allowed us to tune various aspects of the matrix system such as the gelation rates, biodegradability, rheological properties, and biofunctionality. The hydrogels can encapsulate cells and support cell survival.

Detailed analysis of the DNA recognition motifs of the Xanthomonas type III effectors AvrBs3 and AvrBs3Deltarep16.

The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) employs a type III secretion system to translocate effector proteins into plant cells where they modulate host signaling pathways to the pathogen's benefit. The effector protein AvrBs3 acts as a eukaryotic transcription factor and induces the expression of plant genes termed UPA (up-regulated by AvrBs3). Here, we describe 11 new UPA genes from bell pepper that are induced by AvrBs3 early after infection with Xcv. Sequence comparisons revealed the presence of a conserved AvrBs3-responsive element, the UPA box, in all UPA gene promoters analyzed. Analyses of UPA box mutant derivatives confirmed its importance for gene induction by AvrBs3. We show that DNA binding and gene activation were strictly correlated. DNase I footprint studies demonstrated that the UPA box corresponds to the center of the AvrBs3-protected DNA region. Type III delivery of AvrBs3 and mutant derivatives showed that some UPA genes are induced by the AvrBs3 deletion derivative AvrBs3Deltarep16, which lacks four repeats. We show that AvrBs3Deltarep16 recognizes a mutated UPA box with two nucleotide exchanges in positions that are not essential for binding and activation by AvrBs3.

Engineering Hydrogels beyond a Hydrated Network.

In recent years, many mechanical, physical, chemical, and biochemical features of biomatrices have emerged as important properties to dictate the fates of cells. To construct chemically defined biomaterials to recapitulate various biological niches for both cell biology research and therapeutic utilities, it has become increasingly clear that a simple hydrated polymer network would not be able to provide the complex cues and signaling required for many types of cells. The researchers are facing a growing list of mechanophysical and biochemical properties, while each of them could be an important cellular trigger. To include all these design parameters in screening and synthesis is practically difficult, if not impossible. Developing novel high throughput screening technology by combining assay miniaturization, computer simulations, and modeling can help researchers to tackle the challenge to identify the most relevant parameters to tailor materials for specific applications.

Peptidyl prolyl cis/trans isomerase activity on the cell surface correlates with extracellular matrix development.

Interactions with the extracellular matrix (ECM) dictate cell fates. However, the complexity of dense ECM network and cell-surface molecules prevent the study of their dynamic interaction at the molecular level on living cells. Here, we focus on peptidyl prolyl cis/trans isomerases (PPIases) to dissect prolyl isomerization from other dynamic events. We reveal the contribution of PPIase on the mechanical properties of various ECM materials and on the dynamic cell-ECM interaction. To avoid complications associated with the existing spectroscopy-based methods such as light scattering, an assay was developed for detecting PPIase activity on living cell surface. This assay allows us to correlate PPIase activity with ECM development, and with the physiological and pathological states of the cells, including the functional properties of cancer cells and immune effector cells.

Cytocompatible, Injectable, and Electroconductive Soft Adhesives with Hybrid Covalent/Noncovalent Dynamic Network.

Synthetic conductive biopolymers have gained increasing interest in tissue engineering, as they can provide a chemically defined electroconductive and biomimetic microenvironment for cells. In addition to low cytotoxicity and high biocompatibility, injectability and adhesiveness are important for many biomedical applications but have proven to be very challenging. Recent results show that fascinating material properties can be realized with a bioinspired hybrid network, especially through the synergy between irreversible covalent crosslinking and reversible noncovalent self-assembly. Herein, a polysaccharide-based conductive hydrogel crosslinked through noncovalent and reversible covalent reactions is reported. The hybrid material exhibits rheological properties associated with dynamic networks such as self-healing and stress relaxation. Moreover, through fine-tuning the network dynamics by varying covalent/noncovalent crosslinking content and incorporating electroconductive polymers, the resulting materials exhibit electroconductivity and reliable adhesive strength, at a similar range to that of clinically used fibrin glue. The conductive soft adhesives exhibit high cytocompatibility in 2D/3D cell cultures and can promote myogenic differentiation of myoblast cells. The heparin-containing electroconductive adhesive shows high biocompatibility in immunocompetent mice, both for topical application and as injectable materials. The materials could have utilities in many biomedical applications, especially in the area of cardiovascular diseases and wound dressing.

Assembling and decorating hyaluronan hydrogels with twin protein superglues to mimic cell-cell interactions.

Simple polymeric scaffolds have yielded dramatic effects on cell behavior. For more sophisticated phenotypes, precise and efficient chemistries are desired to incorporate proteins into these scaffolds. Here we derivatize hyaluronan with an elastin-like polypeptide containing telechelic SpyTags (HA-SpyTag). Our second network component, the TriCatcher protein, had two SpyCatchers and a terminal SnoopCatcher. Mixing HA-SpyTag with TriCatcher led to rapid hydrogel formation, via spontaneous amidation. SnoopCatcher allowed modular network decoration with SnoopTagJr-linked adhesion molecules, through orthogonal transamidation. This programmed scaffold enables the testing of how individual matrix-anchored protein interactions affect cell behavior. Epithelial cell adhesion molecule (EpCAM) regulates cell behavior and migration, with important effects in cancer. EpCAM-anchoring to the hydrogel induced disassembly of non-malignant mammary spheres in 3D culture. Integrating signaling proteins into biomaterials via efficient biocompatible chemistry should reveal key cues to control cell behavior.

Layer-by-Layer Assembly of Heparin and Peptide-Polyethylene Glycol Conjugates to Form Hybrid Nanothin Films of Biomatrices.

We investigated the utility of a heparin/peptide-polyethylene glycol conjugate system to build layer-by-layer (LbL) structures, to assemble tailored multilayer-biomatrices for cell culture. The LbL assembly balances the advantages of polyelectrolyte systems and protein-based systems. Human umbilical vein endothelial cells showed distinct responses to the film thickness and structure; the presence, density, and spatial arrangement of a cell adhesion ligand within the nanothin film; and the pretreatment of the film with morphogens. The LbL technique presents a versatile tool for modifying cell culture substrates with defined and diverse biochemical and structural features, for investigating cell-material interactions.

Coacervation-Mediated Combinatorial Synthesis of Biomatrices for Stem Cell Culture and Directed Differentiation.

Combinatorial screening represents a promising strategy to discover biomaterials for tailored cell culture applications. Although libraries incorporating different biochemical cues have been investigated, few simultaneously recapitulate relevant biochemical, physical, and dynamic features of the extracellular matrix (ECM). Here, a noncovalent system based on liquid-liquid phase separation (coacervation) and gelation mediated by glycosaminoglycan (GAG)-peptide interactions is reported. Multiple biomaterial libraries are generated using combinations of sulfated glycosaminoglycans and poly(ethylene glycol)-conjugated peptides. Screening these biomaterials reveals preferred biomatrices for the attachment of six cell types, including primary mesenchymal stromal cells (MSCs) and primary neural precursor cells (NPCs). Incorporation of GAGs sustains the expansion of all tested cell types comparable to standard cell culture surfaces, while osteogenic differentiation of MSC and neuronal differentiation of NPC are promoted on chondroitin and heparan biomatrices, respectively. The presented noncovalent system provides a powerful tool for developing tissue-specific ECM mimics.