Intracellular immune sensing promotes inflammation via gasdermin D-driven release of a lectin alarmin.

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

Programming co-assembled peptide nanofiber morphology via anionic amino acid type: Insights from molecular dynamics simulations.

Co-assembling peptides can be crafted into supramolecular biomaterials for use in biotechnological applications, such as cell culture scaffolds, drug delivery, biosensors, and tissue engineering. Peptide co-assembly refers to the spontaneous organization of two different peptides into a supramolecular architecture. Here we use molecular dynamics simulations to quantify the effect of anionic amino acid type on co-assembly dynamics and nanofiber structure in binary CATCH(+/-) peptide systems. CATCH peptide sequences follow a general pattern: CQCFCFCFCQC, where all C's are either a positively charged or a negatively charged amino acid. Specifically, we investigate the effect of substituting aspartic acid residues for the glutamic acid residues in the established CATCH(6E-) molecule, while keeping CATCH(6K+) unchanged. Our results show that structures consisting of CATCH(6K+) and CATCH(6D-) form flatter ß-sheets, have stronger interactions between charged residues on opposing ß-sheet faces, and have slower co-assembly kinetics than structures consisting of CATCH(6K+) and CATCH(6E-). Knowledge of the effect of sidechain type on assembly dynamics and fibrillar structure can help guide the development of advanced biomaterials and grant insight into sequence-to-structure relationships.

Physical tuning of galectin-3 signaling.

Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3-glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits ("Dimer," "Trimer," "Tetramer," "Pentamer," "Hexamer") demonstrated glycan-binding properties and cell death-inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

Anatomy of a selectively coassembled ß-sheet peptide nanofiber.

Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar ß-sheet structures, is common in nature and widely used to fabricate synthetic biomaterials. Selective coassembly of peptide pairs A and B with complementary partial charges is gaining interest due to its potential for expanding the form and function of biomaterials that can be realized. It has been hypothesized that charge-complementary peptides organize into alternating ABAB-type arrangements within assembled ß-sheets, but no direct molecular-level evidence exists to support this interpretation. We report a computational and experimental approach to characterize molecular-level organization of the established peptide pair, CATCH. Discontinuous molecular dynamics simulations predict that CATCH(+) and CATCH(-) peptides coassemble but do not self-assemble. Two-layer ß-sheet amyloid structures predominate, but off-pathway ß-barrel oligomers are also predicted. At low concentration, transmission electron microscopy and dynamic light scattering identified nonfibrillar ∼20-nm oligomers, while at high concentrations elongated fibers predominated. Thioflavin T fluorimetry estimates rapid and near-stoichiometric coassembly of CATCH(+) and CATCH(-) at concentrations ≥100 µM. Natural abundance 13C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(-) coassemble into two-component nanofibers instead of self-sorting. However, 13C-13C dipolar recoupling solid-state NMR measurements also identify nonnegligible AA and BB interactions among a majority of AB pairs. Collectively, these results demonstrate that strictly alternating arrangements of ß-strands predominate in coassembled CATCH structures, but deviations from perfect alternation occur. Off-pathway ß-barrel oligomers are also suggested to occur in coassembled ß-strand peptide systems.

Side-Chain Chemistry Governs Hierarchical Order of Charge-Complementary ß-sheet Peptide Coassemblies.

Self-assembly of proteinaceous biomolecules into functional materials with ordered structures that span length scales is common in nature yet remains a challenge with designer peptides under ambient conditions. This report demonstrates how charged side-chain chemistry affects the hierarchical co-assembly of a family of charge-complementary ß-sheet-forming peptide pairs known as CATCH(X+/Y-) at physiologic pH and ionic strength in water. In a concentration-dependent manner, the CATCH(6K+) (Ac-KQKFKFKFKQK-Am) and CATCH(6D-) (Ac-DQDFDFDFDQD-Am) pair formed either ß-sheet-rich microspheres or ß-sheet-rich gels with a micron-scale plate-like morphology, which were not observed with other CATCH(X+/Y-) pairs. This hierarchical order was disrupted by replacing D with E, which increased fibril twisting. Replacing K with R, or mutating the N- and C-terminal amino acids in CATCH(6K+) and CATCH(6D-) to Qs, increased observed co-assembly kinetics, which also disrupted hierarchical order. Due to the ambient assembly conditions, active CATCH(6K+)-green fluorescent protein fusions could be incorporated into the ß-sheet plates and microspheres formed by the CATCH(6K+/6D-) pair, demonstrating the potential to endow functionality.

Characterizing the Physical Properties and Cell Compatibility of Phytoglycogen Extracted from Different Sweet Corn Varieties.

Owing to its unique structure and properties, the glucose dendrimer phytoglycogen is gaining interest for medical and biotechnology applications. Although many maize variants are available from commercial and academic breeding programs, most applications rely on phytoglycogen extracted from the common maize variant, sugary1. Here we characterized the solubility, hydrodynamic diameter, water-binding properties, protein contaminant concentration, and cytotoxicity of phytoglycogens from different maize sources, A632su1, A619su1, Wesu7, and Ia453su1, harboring various sugary1 mutants. A619su1-SW phytoglycogen was cytotoxic while A632su1-SW phytoglycogen was not. A632su1-Pu phytoglycogen promoted cell growth, whereas extracts from A632su1-NE, A632su1-NC, and A632su1-CM were cytotoxic. Phytoglycogen extracted from Wesu7su1-NE using ethanol precipitation was cytotoxic. Acid-treatment improved Wesu7 phytoglycogen cytocompatibility. Protease-treated Wesu7 extracts promoted cell growth. Phytoglycogen extracted from Ia453su1 21 days after pollination ("Ia435su1 21DAP") was cytotoxic, whereas phytoglycogen extracted at 40 days ("Ia435su1 40DAP") was not. In general, size and solubility had no correlation with cytocompatibility, whereas protein contaminant concentration and water-binding properties did. A632su1-CM had the highest protein contamination among A632 mutants, consistent with its higher cytotoxicity. Likewise, Ia435su1 21DAP phytoglycogen had higher protein contamination than Ia435su1 40DAP. Conversely, protease-treated Wesu7 extracts had lower protein contamination than the other Wesu7 extracts. A632su1-NE, A632su1-NC, and A632su1-CM had similar water-binding properties which differed from those of A632su1-Pu and A632su1-SW. Likewise, water binding differed between Ia435su1 21DAP and Ia435su1 40DAP. Collectively, these data demonstrate that maize phytoglycogen extracts are not uniformly cytocompatible. Rather, maize variant, plant genotype, protein contaminants, and water-binding properties are determinants of phytoglycogen cytotoxicity.

Using Self-Assembling Peptides to Integrate Biomolecules into Functional Supramolecular Biomaterials.

Throughout nature, self-assembly gives rise to functional supramolecular biomaterials that can perform complex tasks with extraordinary efficiency and specificity. Inspired by these examples, self-assembly is increasingly used to fabricate synthetic supramolecular biomaterials for diverse applications in biomedicine and biotechnology. Peptides are particularly attractive as building blocks for these materials because they are based on naturally derived amino acids that are biocompatible and biodegradable; they can be synthesized using scalable and cost-effective methods, and their sequence can be tailored to encode formation of diverse architectures. To endow synthetic supramolecular biomaterials with functional capabilities, it is now commonplace to conjugate self-assembling building blocks to molecules having a desired functional property, such as selective recognition of a cell surface receptor or soluble protein, antigenicity, or enzymatic activity. This review surveys recent advances in using self-assembling peptides as handles to incorporate biologically active molecules into supramolecular biomaterials. Particular emphasis is placed on examples of functional nanofibers, nanovesicles, and other nano-scale structures that are fabricated by linking self-assembling peptides to proteins and carbohydrates. Collectively, this review highlights the enormous potential of these approaches to create supramolecular biomaterials with sophisticated functional capabilities that can be finely tuned to meet the needs of downstream applications.

Engineering Reactive Oxygen Species-Resistant Galectin-1 Dimers with Enhanced Lectin Activity.

Galectin-1 is an immunomodulatory carbohydrate-binding protein with demonstrated efficacy in various preclinical models. However, its potential for clinical use is challenged by two features of the protein. First, galectin-1 (Gal-1) can be inactivated in oxidative environments, such as sites of inflammation, via covalent cross-linking of surface-exposed cysteine residues. Second, the active conformation of galectin-1 is a noncovalent homodimer with a micromolar dissociation constant. Together, these features necessitate frequent administration of high doses of galectin-1 for therapeutic efficacy. To address this challenge, we report an engineered dimeric variant of Gal-1 that is resistant to oxidative inactivation. Specifically, to prevent oxidative inactivation we mutated 3 of 4 surface cysteine residues to serine residues on Gal-1 ("Tri Gal-1"), and then cross-linked two Tri Gal-1 molecules with poly(ethylene glycol) diacrylate to create a stable homodimer ("Tri-PEG-Tri"). Our data demonstrate that cysteine-to-serine galectin-1 mutants retain the carbohydrate-binding properties and pro-apoptotic activity of wild-type Gal-1. Mutants lacking all surface cysteine residues are completely resistant to covalent cross-linking in oxidative environments. At sufficient polymer:protein ratios, poly(ethylene glycol) diacrylate reacts with the surface cysteine on two Tri Gal-1 molecules to form Tri-PEG-Tri. The effective dose of Tri-PEG-Tri is more than an order of magnitude lower than that of non-PEGylated Gal-1. Together, these data demonstrate reactive oxygen species (ROS)-resistant Tri-PEG-Tri dimers with enhanced lectin activity that may be broadly useful for improving the therapeutic efficacy of Gal-1 in immune modulation, transplant tolerance, and treatment of chronic inflammation.

Enzymes as Immunotherapeutics.

Enzymes are attractive as immunotherapeutics because they can catalyze shifts in the local availability of immunostimulatory and immunosuppressive signals. Clinical success of enzyme immunotherapeutics frequently hinges upon achieving sustained biocatalysis over relevant time scales. The time scale and location of biocatalysis are often dictated by the location of the substrate. For example, therapeutic enzymes that convert substrates distributed systemically are typically designed to have a long half-life in circulation, whereas enzymes that convert substrates localized to a specific tissue or cell population can be more effective when designed to accumulate at the target site. This Topical Review surveys approaches to improve enzyme immunotherapeutic efficacy via chemical modification, encapsulation, and immobilization that increases enzyme accumulation at target sites or extends enzyme half-life in circulation. Examples provided illustrate "replacement therapies" to restore deficient enzyme function, as well as "enhancement therapies" that augment native enzyme function via supraphysiologic doses. Existing FDA-approved enzyme immunotherapies are highlighted, followed by discussion of emerging experimental strategies such as those designed to enhance antitumor immunity or resolve inflammation.

Gradated assembly of multiple proteins into supramolecular nanomaterials.

Biomaterials exhibiting precise ratios of different bioactive protein components are critical for applications ranging from vaccines to regenerative medicine, but their design is often hindered by limited choices and cross-reactivity of protein conjugation chemistries. Here, we describe a strategy for inducing multiple different expressed proteins of choice to assemble into nanofibres and gels with exceptional compositional control. The strategy employs 'ßTail' tags, which allow for good protein expression in bacteriological cultures, yet can be induced to co-assemble into nanomaterials when mixed with additional ß-sheet fibrillizing peptides. Multiple different ßTail fusion proteins could be inserted into peptide nanofibres alone or in combination at predictable, smoothly gradated concentrations, providing a simple yet versatile route to install precise combinations of proteins into nanomaterials. The technology is illustrated by achieving precisely targeted hues using mixtures of fluorescent proteins, by creating nanofibres bearing enzymatic activity, and by adjusting antigenic dominance in vaccines.

Immunometabolic Approaches Mitigating Foreign Body Response and Transcriptome Characterization of the Foreign Body Capsule.

Directing immunometabolism presents new opportunities to modulate key cell types associated with the formation of foreign body response (FBR) capsule. Contrasting approaches directing immunometabolism are investigated to mitigate FBR: a broadly suppressive metabolic inhibitor (MI) cocktail comprised of 2-deoxyglucose (2-DG), metformin, and 6-diazo-5-oxo-l-norleucine (DON) with daily systemic dosing regimen, and local weekly injection of the more narrowly focused tryptophan catabolizing IDO-Gal3 fusion protein. Treatments significantly decrease FBR capsule formed around subcutaneously implanted cellulose disks. MI cocktail results in a substantially thinner FBR capsule (40% of control), while weekly local injection of IDO-Gal3 also results in a thinner FBR capsule (69% of control). RNA-sequencing capsule transcripts reveal MI cocktail promotes quiescence, with decreased antigen processing and presentation, T helper subset differentiation, and cytokine-cytokine receptor pathway. IDO-Gal3 promotes pro-regenerative, alternatively activated M2-like macrophages and T helper 2 cells, with increased expression of type 2 response-associated genes (Il4, Il13, Arg1, Mrc1, Chil3, Gata3). IDO-Gal3 decreases pro-inflammatory innate sensing pathways, and C-type lectin receptor, NOD-like receptor, RIG-I-like receptor, and Toll-like receptor signaling. This work helps define key gene targets and pathways concomitantly regulated in the FBR capsule during immunometabolic modulation compared to control FBR capsule.

Parallel ß-Sheet Structure and Structural Heterogeneity Detected within Q11 Self-Assembling Peptide Nanofibers.

Q11 peptide nanofibers are used as a biomaterial for applications such as antigen presentation and tissue engineering, yet detailed knowledge of molecular-level structure has not been reported. The Q11 peptide sequence was designed using heuristics-based patterning of hydrophobic and polar amino acids with oppositely charged amino acids placed at opposite ends of the sequence to promote antiparallel ß-sheet formation. In this work, we employed solid-state nuclear magnetic resonance spectroscopy (NMR) to evaluate whether the molecular organization within Q11 self-assembled peptide nanofibers is consistent with the expectations of the peptide designers. We discovered that Q11 forms a distribution of molecular structures. NMR data from two-dimensional (2D) 13C-13C dipolar-assisted rotational resonance indicate that the K3 and E9 residues between Q11 ß-strands are spatially proximate (within ∼0.6 nm). Frequency-selective rotational echo double resonance (fsREDOR) on K3 Nζ and E9 Cδ-labeled sites showed that approximately 9% of the sites are close enough for salt bridge formation to occur. Surprisingly, dipolar recoupling measurements revealed that Q11 peptides do not assemble into antiparallel ß-sheets as expected, and structural analysis using Fourier-transform infrared spectroscopy and 2D NMR alone can be misleading. 13C PITHIRDS-CT dipolar recoupling measurements showed that the most abundant structure consists of parallel ß-sheets, in contrast to the expected antiparallel ß-sheet structure. Structural heterogeneity was detected from 15N{13C} REDOR measurements, with approximately 22% of ß-strands having antiparallel nearest neighbors. We cannot propose a complete structural model of Q11 nanofibers because of the complexity involved when examining structurally heterogeneous samples using NMR. Altogether, our results show that while heuristics-based patterning is effective in promoting ß-sheet formation, designing a peptide sequence to form a targeted ß-strand arrangement remains challenging.

Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry-state protein storage.

Protein biotherapeutics typically require expensive cold-chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold-chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., "cryoprotection" and "lyoprotection"), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake-forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1-16) were extracted via ethanol precipitation. PG size was generally consistent at ~70-100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1-16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, ß-galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate-buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein-PG13 mixtures had a cake-like appearance and all frozen protein-PG13 mixtures had a Tg' of ~ -26°C. The lyoprotection and cake-forming properties of PG13 were density-dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

Intra-articular delivery of an indoleamine 2,3-dioxygenase galectin-3 fusion protein for osteoarthritis treatment in male Lewis rats.

OBJECTIVE: Osteoarthritis (OA) is driven by low-grade inflammation, and controlling local inflammation may offer symptomatic relief. Here, we developed an indoleamine 2,3-dioxygenase and galectin-3 fusion protein (IDO-Gal3), where IDO increases the production of local anti-inflammatory metabolites and Gal3 binds carbohydrates to extend IDO's joint residence time. In this study, we evaluated IDO-Gal3's ability to alter OA-associated inflammation and pain-related behaviors in a rat model of established knee OA. METHODS: Joint residence was first evaluated with an analog Gal3 fusion protein (NanoLuc™ and Gal3, NL-Gal3) that produces luminescence from furimazine. OA was induced in male Lewis rats via a medial collateral ligament and medial meniscus transection (MCLT + MMT). At 8 weeks, NL or NL-Gal3 were injected intra-articularly (n = 8 per group), and bioluminescence was tracked for 4 weeks. Next, IDO-Gal3s's ability to modulate OA pain and inflammation was assessed. Again, OA was induced via MCLT + MMT in male Lewis rats, with IDO-Gal3 or saline injected into OA-affected knees at 8 weeks post-surgery (n = 7 per group). Gait and tactile sensitivity were then assessed weekly. At 12 weeks, intra-articular levels of IL6, CCL2, and CTXII were assessed. RESULTS: The Gal3 fusion increased joint residence in OA and contralateral knees (p < 0.0001). In OA-affected animals, both saline and IDO-Gal3 improved tactile sensitivity (p = 0.008), but IDO-Gal3 also increased walking velocities (p ≤ 0.033) and improved vertical ground reaction forces (p ≤ 0.04). Finally, IDO-Gal3 decreased intra-articular IL6 levels within the OA-affected joint (p = 0.0025). CONCLUSION: Intra-articular IDO-Gal3 delivery provided long-term modulation of joint inflammation and pain-related behaviors in rats with established OA.

Intra-Articular Delivery of an Indoleamine 2,3-Dioxygenase Galectin-3 Fusion Protein for Osteoarthritis Treatment in Male Lewis Rats.

Objective : Controlling joint inflammation can improve osteoarthritis (OA) symptoms; however, current treatments often fail to provide long-term effects. We have developed an indoleamine 2,3-dioxygenase and galectin-3 fusion protein (IDO-Gal3). IDO converts tryptophan to kynurenines, directing the local environment toward an anti-inflammatory state; Gal3 binds carbohydrates and extends IDO's joint residence time. In this study, we evaluated IDO-Gal3's ability to alter OA-associated inflammation and pain-related behaviors in a rat model of established knee OA. Methods : Joint residence was first evaluated with an analog Gal3 fusion protein (NanoLuc™ and Gal3, NL-Gal3) that produces luminescence from furimazine. OA was induced in male Lewis rats via a medial collateral ligament and medial meniscus transection (MCLT+MMT). At 8 weeks, NL or NL-Gal3 were injected intra-articularly (n=8 per group), and bioluminescence was tracked for 4 weeks. Next, IDO-Gal3's ability to modulate OA pain and inflammation was assessed. Again, OA was induced via MCLT+MMT in male Lewis rats, with IDO-Gal3 or saline injected into OA-affected knees at 8 weeks post-surgery (n=7 per group). Gait and tactile sensitivity were then assessed weekly. At 12 weeks, intra-articular levels of IL6, CCL2, and CTXII were assessed. Results : The Gal3 fusion increased joint residence in OA and contralateral knees (p<0.0001). In OA-affected animals, IDO-Gal3 improved tactile sensitivity (p=0.002), increased walking velocities (p≤0.033), and improved vertical ground reaction forces (p≤0.04). Finally, IDO-Gal3 decreased intra-articular IL6 levels within the OA-affected joint (p=0.0025). Conclusion : Intra-articular IDO-Gal3 delivery provided long-term modulation of joint inflammation and pain-related behaviors in rats with established OA.

Suppression of local inflammation via galectin-anchored indoleamine 2,3-dioxygenase.

The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 (Gal3). Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions.

Heterogeneous protein co-assemblies with tunable functional domain stoichiometry.

In nature, the precise heterogeneous co-assembly of different protein domains gives rise to supramolecular machines that perform complex functions through the co-integrated activity of the individual protein subunits. A synthetic approach capable of mimicking this process would afford access to supramolecular machines with new or improved functional capabilities. Here we show that the distinct peptide strands of a heterotrimeric α-helical coiled-coil (i.e., peptides "A", "B", and "C") can be used as fusion tags for heterogeneous co-assembly of proteins into supramolecular structures with tunable subunit stoichiometry. In particular, we demonstrate that recombinant fusion of A with NanoLuc luciferase (NL-A), B with superfolder green fluorescent protein (sfGFP-B), and C with mRuby (mRuby-C) enables formation of ternary complexes capable of simultaneously emitting blue, green, and red light via sequential bioluminescence and fluorescence resonance energy transfer (BRET/FRET). Fusion of galectin-3 onto the C-terminus of NL-A, sfGFP-B, and mRuby-C endows the ternary complexes with lactose-binding affinity that can be tuned by varying the number of galectin-3 domains integrated into the complex from one to three, while maintaining BRET/FRET function. The modular nature of the fusion protein design, the precise control of domain stoichiometry, and the multiplicity afforded by the three-stranded coiled-coil scaffold provides access to a greater range of subunit combinations than what is possible with heterodimeric coiled-coils used previously. We envision that access to this expanded range of co-integrated protein domain diversity will be advantageous for future development of designer supramolecular machines for therapeutic, diagnostic, and biotechnology applications.

Computational Design and Experimental Validation of ACE2-Derived Peptides as SARS-CoV-2 Receptor Binding Domain Inhibitors.

The COVID-19 pandemic has caused significant social and economic disruption across the globe. Cellular entry of SARS-CoV-2 into the human body is mediated via binding of the Receptor Binding Domain (RBD) on the viral Spike protein (SARS-CoV-2 RBD) to Angiotensin-Converting Enzyme 2 (ACE2) expressed on host cells. Molecules that can disrupt ACE2:RBD interactions are attractive therapeutic candidates to prevent virus entry into human cells. A computational strategy that combines our Peptide Binding Design (PepBD) algorithm with atomistic molecular dynamics simulations was used to design new inhibitory peptide candidates via sequence iteration starting with a 23-mer peptide, referred to as SBP1. SBP1 is derived from a region of the ACE2 Peptidase Domain α1 helix that binds to the SARS-CoV-2 RBD of the initial Wuhan-Hu-1 strain. Three peptides demonstrated a solution-phase RBD-binding dissociation constant in the micromolar range during tryptophan fluorescence quenching experiments, one peptide did not bind, and one was insoluble at micromolar concentrations. However, in competitive ELISA assays, none of these peptides could outcompete ACE2 binding to SARS-CoV-2-RBD up to concentrations of 50 µM, similar to the parent SBP1 peptide which also failed to outcompete ACE2:RBD binding. Molecular dynamics simulations suggest that P4 would have a good binding affinity for the RBD domain of Beta-B.1.351, Gamma-P.1, Kappa-B.1.617.1, Delta-B.1.617.2, and Omicron-B.1.1.529 variants, but not the Alpha variant. Consistent with this, P4 bound Kappa-B.1.617.1 and Delta-B.1.617.2 RBD with micromolar affinity in tryptophan fluorescence quenching experiments. Collectively, these data show that while relatively short unstructured peptides can bind to SARS-CoV-2 RBD with moderate affinity, they are incapable of outcompeting the strong interactions between RBD and ACE2.

Experimental and Computational Models of Transport of Galectin-3 Through Glycosylated Matrix.

Altered extracellular matrix (ECM) production is a hallmark of many fibroproliferative diseases, including certain cancers. The high incidence of glycan-rich components within altered ECM makes the use of glycan-binding proteins such as Galectin-3 (G3) a promising therapeutic strategy. The complexity of ECM as a rich 3D network of proteins with varied glycosylation states makes it challenging to determine the retention of glycan-binding proteins in altered ECM environments. Computational models capable of predicting the transport of glycan-binding proteins in altered ECM can benefit the design and testing of such proteins and associated novel therapeutic strategies. However, such computational models require many kinetic parameters that cannot be estimated from traditional 2D pharmacokinetic assays. To validate transport properties of G3 in 3D ECM constructs, we developed a species transport model that includes diffusion and matrix-binding components to predict retention of G3 fusion proteins in glycan-rich ECM. By iteratively comparing our computational model to experimental results, we are able to determine a reasonable range of parameters for a robust computational model of G3 transport. We anticipate this overall approach to building a data-driven model is translatable to other ECM-targeting therapeutic strategies.

Biomaterials that regulate growth factor activity via bioinspired interactions.

Growth factor activity is localized within the natural extracellular matrix (ECM) by specific non-covalent interactions with core ECM biomolecules, such as proteins and proteoglycans. Recently, these interactions have inspired us and others to develop synthetic biomaterials that can non-covalently regulate growth factor activity for tissue engineering applications. For example, biomaterials covalently or non-covalently modified with heparin glycosaminoglycans can augment growth factor release strategies. In addition, recent studies demonstrate that biomaterials modified with heparin-binding peptides can sequester cell-secreted heparin proteoglycans and, in turn, sequester growth factors and regulate stem cell behavior. Another set of studies show that modular versions of growth factor molecules can be designed to interact with specific components of natural and synthetic ECMs, including collagen and hydroxyapatite. In addition, layer-by-layer assemblies of GAGs and other natural polyelectrolytes retain growth factors at a cell-material interface via specific non-covalent interactions. This review will detail the various bioinspired strategies being used to non-covalently localize growth factor activity within biomaterials, and will highlight in vivo examples of the efficacy of these materials to promote tissue regeneration.