Browsing by Author "Silberg, Jonathan J."

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    Accelerating bioengineering using computer vision, automated microscopy, and synthetic biology
    (2022-04-21) Lee, Jihwan; Silberg, Jonathan J.; St-Pierre , Francois
    Engineering biological entities and systems can provide innovative solutions to healthcare, agriculture, environmental, and manufacturing problems. Nucleic acids, proteins, and cells can be repurposed to function as biosensors, reporters, biochemical foundries and genetic circuits. However, engineering biology is slow and challenging, especially when spatial and temporal properties need to be optimized. This is because traditional methods to optimize spatial and temporal properties have extremely low throughput. Also, transient plasmid transfections in mammalian cells produce high expression heterogeneity making biological systems less predictable and challenging to engineer. We overcame these challenges by leveraging recent developments in computer vision, automated microscopy, and synthetic biology. We developed SPOTlight, a versatile high-throughput bioengineering platform that can screen pooled single-cell variants using a microscope. SPOTlight relies on imaging visual phenotypes by automated microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. We used the platform to screen for 3 million cells expressing mutagenesis libraries and identified a bright variant, mGold, the most photostable yellow fluorescent protein reported to date. Separately, to address the problem of large gene expression variability in mammalian cells, we developed a series of plasmid-based circuits called Equalizers. Equalizers can robustly buffer gene expression heterogeneity caused by plasmid dosage variation between individual mammalian cells. We also show that Equalizers ported on a self-replicating episomal plasmid enable the rapid generation of extrachromosomal cell lines with stable and uniform gene expression. Equalizers are a plasmid-based gene expression system that can facilitate bioengineering in mammalian cells. We believe SPOTlight and Equalizers will enable the rapid engineering of beneficial biological entities and systems that can be deployed to solve pressing problems in many different fields.
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    Artificial Soils Reveal Individual Factor Controls on Microbial Processes
    (American Society for Microbiology, 2022) Del Valle, Ilenne; Gao, Xiaodong; Ghezzehei, Teamrat A.; Silberg, Jonathan J.; Masiello, Caroline A.
    Soil matrix properties influence microbial behaviors that underlie nutrient cycling, greenhouse gas production, and soil formation. However, the dynamic and heterogeneous nature of soils makes it challenging to untangle the effects of different matrix properties on microbial behaviors. To address this challenge, we developed a tunable artificial soil recipe and used these materials to study the abiotic mechanisms driving soil microbial growth and communication. When we used standardized matrices with varying textures to culture gas-reporting biosensors, we found that a Gram-negative bacterium (Escherichia coli) grew best in synthetic silt soils, remaining active over a wide range of soil matric potentials, while a Gram-positive bacterium (Bacillus subtilis) preferred sandy soils, sporulating at low water potentials. Soil texture, mineralogy, and alkalinity all attenuated the bioavailability of an acyl-homoserine lactone (AHL) signaling molecule that controls community-level microbial behaviors. Texture controlled the timing of AHL sensing, while AHL bioavailability was decreased ~105-fold by mineralogy and ~103-fold by alkalinity. Finally, we built artificial soils with a range of complexities that converge on the properties of one Mollisol. As artificial soil complexity increased to more closely resemble the Mollisol, microbial behaviors approached those occurring in the natural soil, with the notable exception of organic matter.
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    Biochar and Microbial Signaling: Production Conditions Determine Effects on Microbial Communication
    (American Chemical Society, 2013) Masiello, Caroline A.; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R.; Rudgers, Jennifer A.; Wagner, Daniel S.; Zygourakis, Kyriacos; Silberg, Jonathan J.
    Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell–cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m2/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m2/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.
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    Charcoal Disrupts Soil Microbial Communication through a Combination of Signal Sorption and Hydrolysis
    (American Chemical Society, 2016) Gao, Xiaodong; Cheng, Hsiao-Ying; Del Valle, Ilenne; Liu, Shirley; Masiello, Caroline A.; Silberg, Jonathan J.
    The presence of charcoal in soil triggers a range of biological effects that are not yet predictable, in part because it interferes with the functioning of chemical signals that microbes release into their environment to communicate. We do not fully understand the mechanisms by which charcoal alters the biologically available concentrations of these intercellular signals. Recently, charcoal has been shown to sorb the signaling molecules that microbes release, rendering them ineffective for intercellular communication. Here, we investigate a second, potentially more important mechanism of interference: signaling-molecule hydrolysis driven by charcoal-induced soil pH changes. We examined the effects of 10 charcoals on the bioavailable concentration of an acyl-homoserine lactone (AHL) used by many Gram-negative bacteria for cell–cell communication. We show that charcoals decrease the level of bioavailable AHL through sorption and pH-dependent hydrolysis of the lactone ring. We then built a quantitative model that predicts the half-lives of different microbial signaling compounds in the presence of charcoals varying in pH and surface area. Our model results suggest that the chemical effects of charcoal on pH-sensitive bacterial AHL signals will be fundamentally distinct from effects on pH-insensitive fungal signals, potentially leading to shifts in microbial community structures.
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    Circular permutation profiling by deep sequencing libraries created using transposon mutagenesis
    (Oxford University Press, 2018) Atkinson, Joshua T.; Jones, Alicia M.; Zhou, Quan; Silberg, Jonathan J.
    Deep mutational scanning has been used to create high-resolution DNA sequence maps that illustrate the functional consequences of large numbers of point mutations. However, this approach has not yet been applied to libraries of genes created by random circular permutation, an engineering strategy that is used to create open reading frames that express proteins with altered contact order. We describe a new method, termed circular permutation profiling with DNA sequencing (CPP-seq), which combines a one-step transposon mutagenesis protocol for creating libraries with a functional selection, deep sequencing and computational analysis to obtain unbiased insight into a protein's tolerance to circular permutation. Application of this method to an adenylate kinase revealed that CPP-seq creates two types of vectors encoding each circularly permuted gene, which differ in their ability to express proteins. Functional selection of this library revealed that >65% of the sampled vectors that express proteins are enriched relative to those that cannot translate proteins. Mapping enriched sequences onto structure revealed that the mobile AMP binding and rigid core domains display greater tolerance to backbone fragmentation than the mobile lid domain, illustrating how CPP-seq can be used to relate a protein's biophysical characteristics to the retention of activity upon permutation.
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    Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions
    (American Chemical Society, 2015) Pandey, Naresh; Nobles, Christopher L.; Zechiedrich, Lynn; Maresso, Anthony W.; Silberg, Jonathan J.
    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a proteinﾖprotein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and proteinﾖprotein interactions within whole animals.
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    Controlling bioenergetic systems using protein design and synthetic biology.
    (2019-03-27) Atkinson, Joshua T; Silberg, Jonathan J.; Bennett, George N.
    An understanding of the mechanisms that life uses to regulate this flow of energy and how to program them at different scales is becoming of great importance for the field of synthetic biology as researches build living systems with ever increasing complexity. My thesis goals are to determine design rules for programming the function of proteins that control energy charge and electron transfer in cells. Herein, I describe my efforts in developing a computational pipeline for analyzing sequencing data from bacterial growth selections that depend on the function of adenylate kinase, a protein that controls cellular energy charge, applying this pipeline to libraries of topological mutants to uncover trends in how the energetic frustration in an allosteric domain relates to tolerance to increased local conformational entropy, developing a high-throughput growth selection for monitoring the efficiency of an electron transfer pathway in vivo, design of synthetic allosteric metalloprotein switches to control electron transfer in the cytosol of cells, and finally coupling cytosolic metabolism to a synthetic extracellular respiratory circuit that enables the transfer of intracellular electrons to surface of cells for reduction of conductive materials. These studies help enable synthetic biology strategies for the control of bioenergetics across a variety of length scales including from local energetics of protein structures to the energy charge of the cell to the energetic interface of cells and materials.
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    Development and Characterization of Split Methyl Halide Transferase for Biosensor Applications in Soil
    (2019-09-10) Huh, Dongkuk; Silberg, Jonathan J.; Bennett, George N.
    Soil microorganisms contribute to agricultural productivity by forming symbiosis with plant roots and affecting the solubility of soil nutrients. Microbiological activity in soil can be monitored directly by coupling the expression of a methyl halide transferase (MHT) to a conditional reporter. This unique bioreporter can provide information about microbial sensing and behaviors without disrupting soil. However, MHT reporting has only been used to report on slow transcriptional processes, limiting its utility. To overcome this challenge, I have created a split MHT (SMHT) that consists of two MHT fragments that are only functional when reconstituted. This SMHT was rationally designed using family sequence information and shown to exhibit MHT-fragment complementation upon fusion to different pairs of interacting proteins. Furthermore, a rapamycin biosensor was constructed that links the detection of rapamycin, a secondary metabolite synthesized by a soil microorganism, to SMHT fragment complementation. The SMHT should be generally useful for reporting on conditional protein-protein interactions in hard-to-image soils and sediments.
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    Effect of protein thermostability on the cooperative function of split enzymes
    (2010) Nguyen, Peter Q.; Silberg, Jonathan J.
    Although the effects of mutational events on protein function cannot yet be predicted a priori, molecular evolution studies have shown that the tolerance of protein structure and function to random mutation is positively correlated with the thermostability of the protein mutated. To test whether thermostability also influences protein function upon random fission, I have characterized the function of split Bacillus subtilis and Thermotoga neapolitana adenylate kinases (AK Bs and AKTn, respectively), enzymes that are required to maintain adenine energy charge. Using libraries of split AKBs and AKTn variants, I show that mesophilic and thermophilic AK orthologs can be split at multiple sites into fragments that complement the growth of Escherichia coli with a temperature-sensitive AK at 40°C. However, I find that the fraction of split AKTn variants that function is ∼7-fold higher than that observed for split AKBs variants. I also find that AKTn can be split within the AMP-binding and LID domains to create functional variants, whereas AKBs can only be split within the AMP-binding domain. Biochemical and biophysical analysis of one pair of homologous split AK variants reveal that polypeptide fragments derived from the more thermostable AK exhibit greater secondary structure and enzymatic activity, suggesting that residual structure of these fragments could account for their retention in function. In addition, complementation studies show that the association and cooperative function of AKBs fragments with little residual structure can be increased by fusing these peptides to interacting proteins. Similarly, the interaction of a split AK Tn can be enhanced at a temperature (78°C) where the fragments are non-functional by fusion to proteins that interact. This split AKTn, which represents the first high-temperature protein fragment complementation assay (ht-PCA) for analyzing protein-protein interactions within a living thermophilic bacterium, is capable of detecting predicted interactions among Thermotoga maritima chemotaxis proteins. These findings show that split proteins with varying functions can be rapidly discovered by fragmenting orthologs with a range of thermostabilities. Moreover, the novel ht-PCA described herein will aid in creating genome-wide maps of thermophilic protein-protein interactions, studying the effects of temperature on biomolecular interactions, and engineering oligomeric thermostable nanomaterials.
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    Encryption of Adeno-Associated Virus for Protease-Controlled Gene Therapy
    (2013-09-16) Judd, Justin; Suh, Junghae; Silberg, Jonathan J.; Segatori, Laura
    Gene therapy holds the unprecedented potential to treat disease by manipulating the underlying genetic blueprints of phenotypic behavior. Targeting of gene delivery is essential to achieve specificity for the intended tissue, which is especially critical in cancer gene therapy to avoid destruction of healthy tissue. Adeno-associated virus (AAV) is considered the safest viral vector and, compared to non-viral vectors, offers several advantages: higher efficiency, genetic modification, combinatorial panning, and high monodispersity. Classic viral targeting has focused on engineering ligand-receptor interactions, but many cell surface targets do not support post-binding transduction events. Furthermore, many potential target tissues – such as triple negative breast cancer – may not display a single, unique identifying surface receptor, so new methods of targeting are needed. Alternatively, many pathological states, including most cancers, exhibit upregulation of proteolytic enzymes in the extracellular milieu. The present work describes the development of an AAV platform that has been engineered to activate in response to disease-related proteases. The specificity and sensitivity of these protease-activatable viruses (PAVs) can be tuned to meet the demands of various clinical scenarios, giving the platform some therapeutic versatility. This work represents the first demonstration of a protease-controlled, non-enveloped virus for genetic therapy. These results extend the therapeutic value of AAV, the safest gene vector currently being explored in 73 clinical trials worldwide.
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    Escort protein regulation of the human mitochondrial molecular chaperone mtHsp70
    (2010) Zhai, Peng; Silberg, Jonathan J.
    Mitochondrial Hsp70 molecular chaperones (mtHsp70) are nuclear-encoded proteins that are needed for the import of proteins into the mitochondrial matrix, biogenesis of Fe/S-clusters involved in energy metabolism, and stress response folding. Unlike the Hsp70 homologs found in other subcellular compartments, two of the mtHsp70 homologs in yeast require a specialized Hsp70 escort protein (Hep1) to maintain their solubility and function. To better understand the role that escort proteins play in regulating their cognate chaperones, I have characterized the regulation of human mtHsp70 by the human Hep, an ortholog of Hep1. In my thesis research, I show that human Hep is localized to the mitochondria of tissue culture cells, like mtHsp70. In addition, I demonstrate that human Hep enhances the solubility of mtHsp70 upon overexpression in Escherichia coli through an interaction with its N-terminal ATPase domain (70ATPase). Chromatography, fluorescence, and copurification analysis using recombinant proteins indicate that Hep binds most tightly to nucleotide-free mtHsp70 and 70ATPase. While this complex is destabilized by the presence of ADP and ATP, ATPase measurements indicate that Hep binds to ATP-bound mtHsp70 (and 70ATPase) and stimulates the steady-state rate of ATP hydrolysis, implicating a role for Hep in directly regulating the mtHsp70 chaperone reaction cycle. To identify Hep residues that are critical for chaperone regulation, I also carried out an alanine mutagenesis scan of charged residues in a tryptophan-free mutant (W1151) of human Hep and assessed the effect of each mutation on Hep and mtHsp70 interactions. In vitro binding studies identified three mutations (R81A, H107A, and D111A) with decreased affinity to nucleotide-free chaperone, and ATPase measurements revealed that one of these mutants (H107A) fails to elicit an increase in the steady-state as activity of 70ATPase. Yeast complementation studies further revealed that Hep supports the growth of Deltahep1 Saccharomyces cerevisiae like yeast Hep1, whereas a Hep-H107A cannot complement Deltahep1 yeast. These findings demonstrate that human Hep is a functional ortholog of yeast Hep1, and provide the first evidence that escort proteins directly regulate the catalytic activity of nucleotide-bound chaperones. Furthermore, they identify a histidine conserved in all mitochondrial and plastid escort proteins as critical for the regulation of human mtHsp70.
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    Evolutionary Relationships Between Low Potential Ferredoxin and Flavodoxin Electron Carriers
    (Frontiers, 2019) Campbell, Ian J.; Bennett, George N.; Silberg, Jonathan J.
    Proteins from the ferredoxin (Fd) and flavodoxin (Fld) families function as low potential electrical transfer hubs in cells, at times mediating electron transfer between overlapping sets of oxidoreductases. To better understand protein electron carrier (PEC) use across the domains of life, we evaluated the distribution of genes encoding [4Fe-4S] Fd, [2Fe-2S] Fd, and Fld electron carriers in over 7,000 organisms. Our analysis targeted genes encoding small PEC genes encoding proteins having ≤200 residues. We find that the average number of small PEC genes per Archaea (~13), Bacteria (~8), and Eukarya (~3) genome varies, with some organisms containing as many as 54 total PEC genes. Organisms fall into three groups, including those lacking genes encoding low potential PECs (3%), specialists with a single PEC gene type (20%), and generalists that utilize multiple PEC types (77%). Mapping PEC gene usage onto an evolutionary tree highlights the prevalence of [4Fe-4S] Fds in ancient organisms that are deeply rooted, the expansion of [2Fe-2S] Fds with the advent of photosynthesis and a concomitant decrease in [4Fe-4S] Fds, and the expansion of Flds in organisms that inhabit low-iron host environments. Surprisingly, [4Fe-4S] Fds present a similar abundance in aerobes as [2Fe-2S] Fds. This bioinformatic study highlights understudied PECs whose structure, stability, and partner specificity should be further characterized.
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    Flavodoxin protein electron carriers: bioinformatic analysis and interactions with sulfite reductases
    (2020-08-14) Guseva, Anna; Silberg, Jonathan J.; Bennett, George N.; Ajo-Franklin, Caroline M.
    Flavodoxins (Flds) are oxidoreductases that distribute electrons to different metabolic pathways through interactions with an array of partner proteins. The aim of my thesis is to understand Fld evolution, establish whether Flds are encoded within the same genomes as Fd-dependent sulfite reductases (SIRs), and demonstrate that a cellular assay can monitor Fld electron transfer (ET) to SIRs. Using bioinformatics, I identify numerous microbes whose genomes encode both Fld and SIR genes. Additionally, I show that Flds can support ET to SIR using a synthetic pathway where protein-mediated ET is monitored using the growth of an Escherichia coli auxotroph that depends upon Fld transferring electrons from a Fd:NADP+ reductase to SIR. My results represent the first evidence that Flds support ET to assimilatory SIRs. Additionally, they show how a synthetic ET pathway in cells can be leveraged to rapidly compare the ET efficiencies of different Flds.
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    Nitrogen, biochar, and mycorrhizae: Alteration of the symbiosis and oxidation of the char surface
    (Elsevier, 2014) LeCroy, Chase; Masiello, Caroline A.; Rudgers, Jennifer A.; Hockaday, William C.; Silberg, Jonathan J.
    In some cases amending soil with biochar improves fertility, although the exact mechanisms through which biochar alters soil processes are not well understood. In other cases, however, biochar amendment can have no effect on plant growth, or can have negative effects. When crop benefits occur, simultaneous amendment with biochar and mineral nutrients causes results that are not additive, suggesting that biochar may be capable of improving the efficiency of nutrient uptake by plants, but the mechanisms of this synergy remain unknown. One possible mechanism that has not been fully explored is alterations to the plant-mycorrhizal fungus mutualism, a relationship that occurs in most land plants. In a 4 week greenhouse experiment, we investigated possible effects of the presence of biochar, mycorrhizal fungi, and nitrogen fertilizer on sorghum seedling growth. Results indicated that the combined treatment of biochar, mycorrhizal fungi, and high nitrogen decreased aboveground plant biomass by 42% relative to the mycorrhizae and high nitrogen treatment, while simultaneously promoting mycorrhizal root colonization. This is evidence for an induced parasitism of the mycorrhizal fungus in the presence of nitrogen and biochar within the 4 week timescale of our experiments. Using x-ray photoelectron spectroscopy, we found evidence of increased surface oxidation on biochar particles over the 4 weeks of our trial, consistent with sorption of labile, plant derived dissolved organic matter or char oxidation, either via biotic or abiotic processes. Biochar in soils with mycorrhizae but without sufficient nitrogen showed more surface oxidation than other treatment combinations, and showed a significantly greater fraction of surface carbon present in carbonyl (ﾖCdouble bond; length as m-dashO) functionalities. Our results suggest that soil nitrogen acts as a switch controlling the ability of char to influence the mycorrhizal symbiosis and, in turn, the degree to which the fungi oxidize the char surface.
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    p38a/Mapkapk2a signaling regulates tristetraprolin in the yolk syncytial layer: A role for mRNA degradation in the morphogenesis of a novel embryonic structure in vertebrate development
    (2014-04-02) Gomez de la Torre Canny, Sol; Wagner, Daniel S.; Bartel, Bonnie; Diehl, Michael R.; Gustin, Michael C.; Silberg, Jonathan J.
    The yolk syncytial layer (YSL) is a novel embryonic structure that is unique to teleost fishes like the zebrafish. How existing genetic mechanisms can change to contribute to the generation of morphological novelties such as the YSL is a fundamental question of evolutionary biology. To address this question we examined the function of mapkapk2a (mk2a). Mk2a is required for YSL morphogenesis. To study the requirement of Mk2a signaling during embryogenesis, we analyzed the betty boop mutant (bbp). Bbp encodes Mk2a, the zebrafish homolog of mammalian MK2, a protein kinase activated by the p38 MAPK signaling pathway. bbp mutants display a striking lysis phenotype. bbp mutant embryos lose the expression of multiple YSL-specific genes. Thus, we examined the role of tristetraprolin (Ttp), a MK2 regulated mRNA-binding protein that promotes degradation of specific mRNA targets. Manipulation of the endogenous activity of Ttp showed that Ttp regulates the stability of YSL-specific mRNA molecules, most notably of mxtx2, which encodes for a zebrafish-specific transcription factor that activates a large proportion of YSL-specific genes. Specific activation of the Mk2a in the YSL inhibits Ttp activity in this cell layer, and prevents expression of Mxtx2 in other cells of the embryo. Expression of Mxtx2 or activation of the p38a /Mk2a pathway outside of the YSL results in dramatic defects in development. MK2 is not required for embryogenesis in mammals. Mutation of MK2 results in impaired inflammatory response and resistance to inflammatory diseases. The ability to manipulate the activity of the members of this conserved pathway in this novel context suggests that epiboly may be a useful platform to probe the molecular mechanism of TTP-dependent mRNA degradation that plays a crucial role in the regulation of the inflammatory response in mammals.
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    Parallelizing Interproscan with SLURM
    (Rice University, 2023-06-27) Fulk, Emily M.; Goldman, Annelise L.; Momper, Lily; Heider, Clinton; Mulligan, John; Osburn, Magdalena; Masiello, Caroline A.; Silberg, Jonathan J.; Systems, Synthetic, and Physical Biology
    This repository contains code to facilitate analysis of large numbers of proteins by Interproscan with minimum user oversight and efficient use of computational resources. It provides templates for parallelizing Interproscan with SLURM on the Rice NOTS computing cluster, following a high-throughput computing model.
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    Random Insertion of mCherry Into VP3 Domain of Adeno- associated Virus Yields Fluorescent Capsids With no Loss of Infectivity
    (American Society of Gene & Cell Therapy, 2012) Judd, Justin; Wei, Fang; Nguyen, Peter Q.; Tartaglia, Lawrence J.; Agbandje-McKenna, Mavis; Silberg, Jonathan J.; Suh, Junghae
    Adeno-associated virus (AAV)-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase). The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.
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    Reprogramming the proteostasis network to prevent the accumulation of alpha-synuclein aggregates
    (2014-03-14) Kilpatrick, Kiri; Segatori, Laura; Gonzalez, Ramon; Silberg, Jonathan J.; Nagrath, Deepak
    Protein misfolding and aggregation characterizes the development of a number of neurodegenerative diseases, such as Parkinson’s, Alzheimer’s and Huntington’s disease. The hallmark of Parkinson’s disease is the formation of proteinaceous inclusions, which consist primarily of α-synuclein (α-syn), a natively unstructured protein with propensity to misfold and aggregate. Cells have evolved sophisticated systems of protein quality control to prevent accumulation of non-native proteins and maintain protein homeostasis. However, the load of misfolded α-syn typically exceeds the capacity of the quality control system. Aberrant accumulation of misfolded α-syn leads to proteotoxic stress, eventually resulting in neurodegeneration. The objective of this project is to investigate chemical and genetic approaches to modulate the protein quality control system and reduce the accumulation of aberrant α-syn species. Studying α-syn aggregation in cells presents a number of challenges mainly due to the limited availability of tools to quantitatively distinguish between different α-syn conformational species within the cellular environment. To address this need, we engineered an in vitro model system based on neuroglioma cells that accumulate α-syn aggregates and developed a set of analytical tools based on the use of aggregation responsive probes to quantify α-syn aggregation in cells. To test whether modulating the protein quality control system affects the accumulation of α-syn aggregates, we investigated a series of complementary approaches aimed at i) enhancing the innate cellular chaperone machinery, which promotes folding and prevents aggregation, and ii) upregulating the autophagy pathway, which mediates clearance of aggregated proteins. We demonstrated that chemical modulation of Hsp70, a ubiquitously expressed molecular chaperone, affects the accumulation of α-syn aggregates. Particularly, the Hsp70 upregulator carbenoxolone was found to reduce α-syn aggregation and prevent α-syn-induced cytotoxicity via activation of the heat shock response. We also found that activation of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, results in enhanced autophagic clearance of α-syn aggregates. We demonstrated that cell treatment with 2-hydroxypropyl-β-cyclodextrin reduces the accumulation of aggregated α-syn specifically by upregulating TFEB-mediated autophagic clearance. These findings lay the foundation for the development of pharmacological strategies to reduce the accumulation of misfolded and aggregated α-syn for the treatment of Parkinson’s disease.
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    SCHEMA Computational Design of Virus Capsid Chimeras: Calibrating How Genome Packaging, Protection, and Transduction Correlate with Calculated Structural Disruption
    (American Chemical Society, 2013) Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae
    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.
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    Solution-Deposited and Patternable Conductive Polymer Thin-Film Electrodes for Microbial Bioelectronics
    (Wiley, 2022) Tseng, Chia-Ping; Liu, Fangxin; Zhang, Xu; Huang, Po-Chun; Campbell, Ian; Li, Yilin; Atkinson, Joshua T.; Terlier, Tanguy; Ajo-Franklin, Caroline M.; Silberg, Jonathan J.; Verduzco, Rafael
    Microbial bioelectronic devices integrate naturally occurring or synthetically engineered electroactive microbes with microelectronics. These devices have a broad range of potential applications, but engineering the biotic–abiotic interface for biocompatibility, adhesion, electron transfer, and maximum surface area remains a challenge. Prior approaches to interface modification lack simple processability, the ability to pattern the materials, and/or a significant enhancement in currents. Here, a novel conductive polymer coating that significantly enhances current densities relative to unmodified electrodes in microbial bioelectronics is reported. The coating is based on a blend of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) crosslinked with poly(2-hydroxyethylacrylate) (PHEA) along with a thin polydopamine (PDA) layer for adhesion to an underlying indium tin oxide (ITO) electrode. When used as an interface layer with the current-producing bacterium Shewanella oneidensis MR-1, this material produces a 178-fold increase in the current density compared to unmodified electrodes, a current gain that is higher than previously reported thin-film 2D coatings and 3D conductive polymer coatings. The chemistry, morphology, and electronic properties of the coatings are characterized and the implementation of these coated electrodes for use in microbial fuel cells, multiplexed bioelectronic devices, and organic electrochemical transistor based microbial sensors are demonstrated. It is envisioned that this simple coating will advance the development of microbial bioelectronic devices.