Templated Synthesis of Copper Nanoclusters with a Hybrid Lysozyme-Polymer Material for Enhanced Fluorescence.

Hybrid materials that combine organic polymers and biomacromolecules offer unique opportunities for precisely controlling 3D chemical environments. Although biological or organic templates have been separately used to control the growth of inorganic nanoclusters, hybrid structures represent a relatively unexplored approach to tailoring nanocluster properties. Here, we demonstrate that a molecularly defined lysozyme-polymer resin material acts as a structural scaffold for the synthesis of copper nanoclusters (CuNCs) with well controlled size distributions. The resulting CuNCs have significantly enhanced fluorescence compared with syntheses based on polymeric or biological templates alone. The synergistic approach described here is appealing for the synthesis of biocompatible fluorescent labels with improved photostability.

Cell factory engineering: Challenges and opportunities for synthetic biology applications.

The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.

Boronic Acid Resin for Selective Immobilization of Canonically Encoded Proteins.

The use of transition-metal-mediated boronic acid chemistry presents a novel method of protein immobilization on a solid support. This is a one-step method that site-selectively immobilizes pyroglutamate-histidine (pGH)-tagged proteins. Herein, we describe the synthesis of alkenylboronic acid-functionalized poly(ethylene glycol) acrylamide (PEGA) resin and its subsequent reactions with pGH-tagged proteins to produce covalent linkages. The selectivity of immobilization is demonstrated within fluorescent studies, model mixtures, and lysates.

Chemical and biological approaches synergize to ameliorate protein-folding diseases.

Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.

A gene signal amplifier platform for monitoring the unfolded protein response.

Gene expression in mammalian cells results from coordinated protein-driven processes guided by diverse mechanisms of regulation, including protein-protein interactions, protein localization, DNA modifications and chromatin rearrangement. Regulation of gene expression is particularly important in stress-response pathways. To address the need to monitor chromosomal gene expression generating a readily detectable signal output that recapitulates gene expression dynamics, we developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal target gene. We generated a multiplex reporter system for monitoring markers of the unfolded protein response, a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene with high sensitivity, this platform provides a technology for monitoring gene expression with superior sensitivity and dynamic resolution.

Open questions: how do engineered nanomaterials affect our cells?

Our cells have evolutionarily conserved mechanisms that battle foreign and toxic materials to maintain cellular homeostasis and viability. How do these cellular machineries respond to engineered nanomaterials?

The importance and future of biochemical engineering.

Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on "The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes". On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

Aggregation Behavior of Nanoparticle-Peptide Systems Affects Autophagy.

The aggregation of nanoparticle colloidal dispersions in complex biological environments changes the nanoparticle properties, such as size and surface area, thus affecting the interaction of nanoparticles at the interface with cellular components and systems. We investigated the effect of nanoparticle aggregation on autophagy, the main catabolic pathway that mediates degradation of nanosized materials and that is activated in response to internalization of foreign nanosized materials. We used carboxylated polystyrene nanoparticles (100 nm) and altered the nanoparticle aggregation behavior through addition of a multidomain peptide, thus generating a set of nanoparticle-peptide mixtures with variable aggregation properties. Specifically, modulating the peptide concentration resulted in nanoparticle-peptide mixtures that are well dispersed extracellularly but aggregate upon cellular internalization. We monitored the effect of internalization of nanoparticle-peptide mixtures on a comprehensive set of markers of the autophagy pathway, ranging from transcriptional regulation to clearance of autophagic substrates. The nanoparticle-peptide mixtures were found to activate the transcription factor EB, a master regulator of autophagy and lysosomal biogenesis. We also found that intracellular aggregation of nanoparticle colloidal dispersions causes blockage of autophagic flux. This study provides important insights on the effect of the aggregation properties of nanoparticles on cells and, particularly, on the main homeostatic pathway activated in response to nanoparticle internalization. These results also point to the need to control the colloidal stability of nanoparticle systems for a variety of biomedical applications.

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan-Lam Coupling.

Manipulation of biomacromolecules is ideally achieved through unique and bioorthogonal chemical reactions of genetically encoded, naturally occurring functional groups. The toolkit of methods for site-specific conjugation is limited by selectivity concerns and a dearth of naturally occurring functional groups with orthogonal reactivity. We report that pyroglutamate amide N-H bonds exhibit bioorthogonal copper-catalyzed Chan-Lam coupling at pyroglutamate-histidine dipeptide sequences. The pyroglutamate residue is readily incorporated into proteins of interest by natural enzymatic pathways, allowing specific bioconjugation at a minimalist dipeptide tag.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

2-Hydroxypropyl-ß-cyclodextrin promotes transcription factor EB-mediated activation of autophagy: implications for therapy.

2-Hydroxypropyl-ß-cyclodextrin (HPßCD) is a Food and Drug Administration-approved excipient used to improve the stability and bioavailability of drugs. Despite its wide use as a drug delivery vehicle and the recent approval of a clinical trial to evaluate its potential for the treatment of a cholesterol storage disorder, the cellular pathways involved in the adaptive response that is activated upon exposure to HPßCD are still poorly defined. Here, we show that cell treatment with HPßCD results in the activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and in enhancement of the cellular autophagic clearance capacity. HPßCD administration promotes transcription factor EB-mediated clearance of proteolipid aggregates that accumulate due to inefficient activity of the lysosome-autophagy system in cells derived from a patient with a lysosomal storage disorder. Interestingly, HPßCD-mediated activation of autophagy was found not to be associated with activation of apoptotic pathways. This study provides a mechanistic understanding of the cellular response to HPßCD treatment, which will inform the development of safe HPßCD-based therapeutic modalities and may enable engineering HPßCD as a platform technology to reduce the accumulation of lysosomal storage material.

TFEB regulates lysosomal proteostasis.

Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a ß-hexosaminidase mutant associated with the development of another LSD, Tay-Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs.

The autophagic response to polystyrene nanoparticles is mediated by transcription factor EB and depends on surface charge.

BACKGROUND: A number of engineered nanoparticles induce autophagy, the main catabolic pathway that regulates bulk degradation of cytoplasmic material by the lysosomes. Depending on the specific physico-chemical properties of the nanomaterial, however, nanoparticle-induced autophagy may have different effects on cell physiology, ranging from enhanced autophagic degradation to blockage of autophagic flux. To investigate the molecular mechanisms underlying the impact of nanoparticle charge on the nature of the autophagic response, we tested polystyrene nanoparticles (50 nm) with neutral, anionic, and cationic surface charges. RESULTS: We found all polystyrene nanoparticles investigated in this study to activate autophagy. We showed that internalization of polystyrene nanoparticles results in activation of the transcription factor EB, a master regulator of autophagy and lysosome biogenesis. Autophagic clearance, however, was observed to depend specifically on the charge of the nanoparticles. Particularly, we found that the autophagic response to polystyrene nanoparticles presenting a neutral or anionic surface involves enhanced clearance of autophagic cargo. Cell exposure to polystyrene nanoparticles presenting a cationic surface, on the other hand, results in transcriptional upregulation of the pathway, but also causes lysosomal dysfunction, ultimately resulting in blockage of autophagic flux. CONCLUSIONS: This study furthers our understanding of the molecular mechanisms that regulate the autophagic response to nanoparticles, thus contributing essential design criteria for engineering benign nanomaterials.

Rapid detection of pathogenic bacteria and screening of phage-derived peptides using microcantilevers.

We report the use of an array of microcantilevers to measure the specific binding of Salmonella to peptides derived from phage display libraries. Selectivity of these phage-derived peptides for Salmonella spp. and other pathogens ( Listeria monocytogenes and Escherichia coli ) are compared with a commercially available anti- Salmonella antibody and the antimicrobial peptide alamethicin. A Langmuir isotherm model was applied to determine the binding affinity constants of the peptides to the pathogens. One particular peptide, MSal 020417, demonstrated a higher binding affinity to Salmonella spp. than the commercially available antibody and is able to distinguish among eight Salmonella serovars on a microcantilever. A multiplexed screening system to quickly determine the binding affinities of various peptides to a particular pathogen highly improves the efficiency of the peptide screening process. Combined with phage-derived peptides, this microcantilever-based technique provides a novel biosensor to rapidly and accurately detect pathogens and holds potential to be further developed as a screening method to identify pathogen-specific recognition elements.

Impairment of homeostasis in lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are inherited metabolic diseases caused by deficiencies in lysosomal proteins, which result in accumulation of undegraded metabolites and disruption of lysosomal proteostasis. Despite significant progress in the molecular genetics and biochemistry underlying the cellular pathogenesis of LSDs, the mechanisms that link accumulation of storage material to development and progression of these diseases are still unclear. At the crossroad of degradative pathways, lysosomes play a fundamental role in the maintenance of cellular homeostasis. Through a series of examples, this review illustrates how defects in lysosomal biogenesis and function impact a number of cellular pathways that are involved in the pathogenic cascade.

Probing the association of triblock copolymers with supported lipid membranes using microcantilevers.

Pluronics are a class of amphiphilic triblock copolymers that are known to interact with cellular membranes in interesting ways. The solubility of these triblock copolymers in free lipid membranes can be altered with temperature, allowing the possibility of tuning their membrane insertion. However, for supported lipid membranes, the asymmetric local environment and the strong influence of the solid support can alter the solubility of these triblock copolymers in lipid membranes. Here, we probe the interactions of these copolymers with supported lipid membranes using microcantilevers and fluorescence recovery after photobleaching (FRAP) measurements. We measure the solubility and interactions of triblock copolymers (F68 and F98) in supported lipid bilayers as a function of temperature and the length of the copolymer lipophilic block. A Langmuir isotherm model and a free mean area theory are applied to describe the polymer-lipid interactions at the microcantilever surface, determine association constants, and analyze the effect of triblock copolymers on lateral lipid diffusion.

A Computational Modeling Approach for the Design of Genetic Control Systems that Respond to Transcriptional Activity.

Recent progress in synthetic biology has enabled the design of complex genetic circuits that interface with innate cellular functions, such as gene transcription, and control user-defined outputs. Implementing these genetic networks in mammalian cells, however, is a cumbersome process that requires several steps of optimization and benefits from the use of predictive modeling. Combining deterministic mathematical models with software-based numerical computing platforms allows researchers to quickly design, evaluate, and optimize multiple circuit topologies to establish experimental constraints that generate the desired control systems. In this chapter, we present a systematic approach based on predictive mathematical modeling to guide the design and construction of gene activity-based sensors. This approach enables user-driven circuit optimization through iterations of sensitivity analyses and parameter scans, providing a universal method to engineer sense and respond cells for diverse applications.

Design of Oscillatory Networks through Post-Translational Control of Network Components.

Many essential functions in biological systems, including cell cycle progression and circadian rhythm regulation, are governed by the periodic behaviors of specific molecules. These periodic behaviors arise from the precise arrangement of components in biomolecular networks that generate oscillatory output signals. The dynamic properties of individual components of these networks, such as maturation delays and degradation rates, often play a key role in determining the network's oscillatory behavior. In this study, we explored the post-translational modulation of network components as a means to generate genetic circuits with oscillatory behaviors and perturb the oscillation features. Specifically, we used the NanoDeg platform-A bifunctional molecule consisting of a target-specific nanobody and a degron tag-to control the degradation rates of the circuit's components and predicted the effect of NanoDeg-mediated post-translational depletion of a key circuit component on the behavior of a series of proto-oscillating network topologies. We modeled the behavior of two main classes of oscillators, namely relaxation oscillator topologies (the activator-repressor and the Goodwin oscillator) and ring oscillator topologies (repressilators). We identified two main mechanisms by which non-oscillating networks could be induced to oscillate through post-translational modulation of network components: an increase in the separation of timescales of network components and mitigation of the leaky expression of network components. These results are in agreement with previous findings describing the effect of timescale separation and mitigation of leaky expression on oscillatory behaviors. This work thus validates the use of tools to control protein degradation rates as a strategy to modulate existing oscillatory signals and construct oscillatory networks. In addition, this study provides the design rules to implement such an approach based on the control of protein degradation rates using the NanoDeg platform, which does not require genetic manipulation of the network components and can be adapted to virtually any cellular protein. This work also establishes a framework to explore the use of tools for post-translational perturbations of biomolecular networks and generates desired behaviors of the network output.

Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.

Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis.

Detection of α-synuclein amyloidogenic aggregates in vitro and in cells using light-switching dipyridophenazine ruthenium(II) complexes.

Protein aggregation is the hallmark of a number of neurodegenerative diseases including Parkinson's and Huntington's diseases. There is a significant interest in understanding the molecular mechanisms involved in the self-association and fibrillization of monomeric soluble proteins into insoluble deposits in vivo and in vitro. Probes with novel properties, such as red-shifted emission, large Stokes shifts, and high photostability, are desirable for a variety of protein aggregation studies. To respond to the increasing need for aggregation-responsive compounds suitable to cellular studies, we present a ruthenium(II) dipyridophenazine derivative, [Ru(phen)(2)dppz](2+) (phen =1,10-phenanthroline, dppz = dipyrido[3,2-a:2'.3'-c]phenazine), to study aggregation of α-synuclein (αS), which is associated with the development of Parkinson's disease. We demonstrated the use of [Ru(phen)(2)dppz](2+) to monitor αS fibril formation in real-time and to detect and quantify αS aggregates in neuroglioma cells, thereby providing a novel molecular tool to study protein deposition diseases in vitro and in vivo.