Genetically encoded lysine photocage for spatiotemporal control of TDP-43 nuclear import.

Intracellular aggregation of transactive response DNA binding protein of 43 kDa (TDP-43) is a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis. While primarily a nuclear protein, TDP-43 translocates to the cytosol during cellular stress. Consequences of cytosolic accumulation of TDP-43 is difficult to evaluate in the absence of exogenous toxins. Here, we demonstrate spatiotemporal control over the nuclear import of TDP-43 by installing a photocage (ortho-nitrobenzyl ester) on a single lysine residue (K84) through amber codon suppression in HEK293T cells. Translocation of this cytosolic construct is photo-triggerable in a dose-dependent manner with 355 nm light. Interestingly, both fluid- and solid-like puncta were found based on fluorescence recovery after photobleaching experiments, similar to what is expected of stress granules and intracellular aggregates, respectively. This optogenetic method is advantageous as it is minimally perturbative and broadly applicable to other studies of protein translocation between cellular compartments.

Versatile Tissue-Injectable Hydrogels with Extended Hydrolytic Release of Bioactive Protein Therapeutics.

Hydrogels generally have broad utilization in healthcare due to their tunable structures, high water content, and inherent biocompatibility. FDA-approved applications of hydrogels include spinal cord regeneration, skin fillers, and local therapeutic delivery. Drawbacks exist in the clinical hydrogel space, largely pertaining to inconsistent therapeutic exposure, short-lived release windows, and difficulties inserting the polymer into tissue. In this study, we engineered injectable, biocompatible hydrogels that function as a local protein therapeutic depot with a high degree of user-customizability. We showcase a PEG-based hydrogel functionalized with bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) handles for its polymerization and functionalization with a variety of payloads. Small-molecule and protein cargos, including chemokines and antibodies, were site-specifically modified with hydrolysable "azidoesters" of varying hydrophobicity via direct chemical conjugation or sortase-mediated transpeptidation. These hydrolysable esters afforded extended release of payloads linked to our hydrogels beyond diffusion; with timescales spanning days to months dependent on ester hydrophobicity. Injected hydrogels polymerize in situ and remain in tissue over extended periods of time. Hydrogel-delivered protein payloads elicit biological activity after being modified with SPAAC-compatible linkers, as demonstrated by the successful recruitment of murine T-cells to a mouse melanoma model by hydrolytically released murine CXCL10. These results highlight a highly versatile, customizable hydrogel-based delivery system for local delivery of protein therapeutics with payload release profiles appropriate for a variety of clinical needs.

The N terminus of α-synuclein dictates fibril formation.

The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson's disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41-140). This behavior can be explained by the unique fibril structure that is adopted by 41-140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent ß-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended ß-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.

Genetically Encoded Photocleavable Linkers for Patterned Protein Release from Biomaterials.

Given the critical role that proteins play in almost all biological processes, there is great interest in controlling their presentation within and release from biomaterials. Despite such outstanding enthusiasm, previously developed strategies in this regard result in ill-defined and heterogeneous populations with substantially decreased activity, precluding their successful application to fragile species including growth factors. Here, we introduce a modular and scalable method for creating monodisperse, genetically encoded chimeras that enable bioactive proteins to be immobilized within and subsequently photoreleased from polymeric hydrogels. Building upon recent developments in chemoenzymatic reactions, bioorthogonal chemistry, and optogenetics, we tether fluorescent proteins, model enzymes, and growth factors site-specifically to gel biomaterials through a photocleavable protein (PhoCl) that undergoes irreversible backbone photoscission upon exposure to cytocompatible visible light (λ ≈ 400 nm) in a dose-dependent manner. Mask-based and laser-scanning lithographic strategies using commonly available light sources are employed to spatiotemporally pattern protein release from hydrogels while retaining their full activity. The photopatterned epidermal growth factor presentation is exploited to promote anisotropic cellular proliferation in 3D. We expect these methods to be broadly useful for applications in diagnostics, drug delivery, and regenerative medicine.

Logic-Based Delivery of Site-Specifically Modified Proteins from Environmentally Responsive Hydrogel Biomaterials.

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though "smart" platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user-specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site-specifically modified proteins from hydrogels following Boolean logic. A sortase-mediated transpeptidation reaction is used to generate recombinant proteins C-terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli-labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two-input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli-sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.

Bioactive site-specifically modified proteins for 4D patterning of gel biomaterials.

Protein-modified biomaterials can be used to modulate cellular function in three dimensions. However, as the dynamic heterogeneous control over complex cell physiology continues to be sought, strategies that permit a reversible and user-defined tethering of fragile proteins to materials remain in great need. Here we introduce a modular and robust semisynthetic approach to reversibly pattern cell-laden hydrogels with site-specifically modified proteins. Exploiting a versatile sortase-mediated transpeptidation, we generate a diverse library of homogeneous, singly functionalized proteins with bioorthogonal reactive handles for biomaterial modification. We demonstrate the photoreversible immobilization of fluorescent proteins, enzymes and growth factors to gels with excellent spatiotemporal resolution while retaining native protein bioactivity. Localized epidermal growth factor presentation enables dynamic regulation over proliferation, intracellular mitogen-activated protein kinase signalling and subcellularly resolved receptor endocytosis. Our method broadly permits the modification and patterning of a wide range of proteins, which provides newfound avenues to probe and direct advanced cellular fates in four dimensions.

Engineered modular biomaterial logic gates for environmentally triggered therapeutic delivery.

The successful transport of drug- and cell-based therapeutics to diseased sites represents a major barrier in the development of clinical therapies. Targeted delivery can be mediated through degradable biomaterial vehicles that utilize disease biomarkers to trigger payload release. Here, we report a modular chemical framework for imparting hydrogels with precise degradative responsiveness by using multiple environmental cues to trigger reactions that operate user-programmable Boolean logic. By specifying the molecular architecture and connectivity of orthogonal stimuli-labile moieties within material cross-linkers, we show selective control over gel dissolution and therapeutic delivery. To illustrate the versatility of this methodology, we synthesized 17 distinct stimuli-responsive materials that collectively yielded all possible YES/OR/AND logic outputs from input combinations involving enzyme, reductant and light. Using these hydrogels we demonstrate the first sequential and environmentally stimulated release of multiple cell lines in well-defined combinations from a material. We expect these platforms will find utility in several diverse fields including drug delivery, diagnostics and regenerative medicine.

Cyclic Stiffness Modulation of Cell-Laden Protein-Polymer Hydrogels in Response to User-Specified Stimuli including Light.

Although mechanical signals presented by the extracellular matrix are known to regulate many essential cell functions, the specific effects of these interactions, particularly in response to dynamic and heterogeneous cues, remain largely unknown. Here, we introduce a modular semisynthetic approach to create protein-polymer hydrogel biomaterials that undergo reversible stiffening in response to user-specified inputs. Employing a novel dual-chemoenzymatic modification strategy, we create fusion protein-based gel crosslinkers that exhibit stimuli-dependent intramolecular association. Linkers based on calmodulin yield calcium-sensitive materials, while those containing the photosensitive LOV2 (light, oxygen, and voltage sensing domain 2) protein give phototunable constructs whose moduli can be cycled on demand with spatiotemporal control about living cells. We exploit these unique materials to demonstrate the significant role that cyclic mechanical loading plays on fibroblast-to-myofibroblast transdifferentiation in three-dimensional (3D) space. Our moduli-switchable materials should prove useful for studies in mechanobiology, providing new avenues to probe and direct matrix-driven changes in 4D cell physiology.

Streamlined Synthesis and Assembly of a Hybrid Sensing Architecture with Solid Binding Proteins and Click Chemistry.

Combining bioorthogonal chemistry with the use of proteins engineered with adhesive and morphogenetic solid-binding peptides is a promising route for synthesizing hybrid materials with the economy and efficiency of living systems. Using optical sensing of chloramphenicol as a proof of concept, we show here that a GFP variant engineered with zinc sulfide and silica-binding peptides on opposite sides of its ß-barrel supports the fabrication of protein-capped ZnS:Mn nanocrystals that exhibit the combined emission signatures of organic and inorganic fluorophores. Conjugation of a chloramphenicol-specific DNA aptamer to the protein shell through strain-promoted azide-alkyne cycloaddition and spontaneous concentration of the resulting nanostructures onto SiO2 particles mediated by the silica-binding sequence enables visual detection of environmentally and clinically relevant concentrations of chloramphenicol through analyte-mediated inner filtering of sub-330 nm excitation light.

Photomediated oxime ligation as a bioorthogonal tool for spatiotemporally-controlled hydrogel formation and modification.

Click chemistry has proved a valuable tool in biocompatible hydrogel formation for 3D cell culture, owing to its bioorthogonal nature and high efficiency under physiological conditions. While traditional click reactions can be readily employed to create uniform functional materials about living cells, their spontaneity prohibits spatiotemporal control of material properties, thereby limiting their utility in recapitulating the dynamic heterogeneity characteristic of the in vivo microenvironment. Photopolymerization-based techniques gain this desired level of 4D programmability, but often at the expense of introducing propagating free radicals that are prone to non-specific reactions with biological systems. Here we present a strategy for bioorthogonal hydrogel formation and modification that does not rely on propagating free radicals, proceeding through oxime ligation moderated by a photocaged alkoxyamine. Upon mild near UV light exposure, the photocage is cleaved, liberating the alkoxyamine and permitting localized condensation with an aldehyde. Multi-arm crosslinkers, functionalized with either benzaldehydes or photocaged alkoxyamines, formed oxime-based hydrogels within minutes of light exposure in the presence of live cells. Polymerization rates and final mechanical properties of these gels could be systematically tuned by varying crosslinker concentrations, light intensity, aniline catalyst equivalents, and pH. Moreover, hydrogel geometry and final mechanical properties were controlled by the location and extent of UV exposure, respectively. Photomediated oxime ligation was then translated to the biochemical modification of hydrogels, where full-length proteins containing photocaged alkoxyamines were immobilized in user-defined regions exposed to UV light. The programmability afforded by photomediated oxime ligation can recapitulate dynamically anisotropic mechanical and biochemical aspects of the native extracellular matrix. Consequently, photopolymerized oxime-based hydrogels are expected to enable an enhanced understanding of cell-matrix interactions by serving as improved 4D cell culture platforms.