4S-fluorination of ProB29 in insulin lispro slows fibril formation.

Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by CD, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.

Quantitative Real-Time Analysis of Living Materials by Stimulated Raman Scattering Microscopy.

Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.

Rare, Tightly-Bound, Multi-Cellular Clusters in the Pancreatic Ducts of Adult Mice Function Like Progenitor Cells and Survive and Proliferate After Acinar Cell Injury.

Pancreatic ductal progenitor cells have been proposed to contribute to adult tissue maintenance and regeneration after injury, but the identity of such ductal cells remains elusive. Here, from adult mice, we identify a near homogenous population of ductal progenitor-like clusters, with an average of 8 cells per cluster. They are a rare subpopulation, about 0.1% of the total pancreatic cells, and can be sorted using a fluorescence-activated cell sorter with the CD133highCD71lowFSCmid-high phenotype. They exhibit properties in self-renewal and tri-lineage differentiation (including endocrine-like cells) in a unique 3-dimensional colony assay system. An in vitro lineage tracing experiment, using a novel HprtDsRed/+ mouse model, demonstrates that a single cell from a cluster clonally gives rise to a colony. Droplet RNAseq analysis demonstrates that these ductal clusters express embryonic multipotent progenitor cell markers Sox9, Pdx1, and Nkx6-1, and genes involved in actin cytoskeleton regulation, inflammation responses, organ development, and cancer. Surprisingly, these ductal clusters resist prolonged trypsin digestion in vitro, preferentially survive in vivo after a severe acinar cell injury and become proliferative within 14 days post-injury. Thus, the ductal clusters are the fundamental units of progenitor-like cells in the adult murine pancreas with implications in diabetes treatment and tumorigenicity.

Incorporation of Aliphatic Proline Residues into Recombinantly Produced Insulin.

Analogs of proline can be used to expand the chemical space about the residue while maintaining its uniquely restricted conformational space. Here, we demonstrate the incorporation of 4R-methylproline, 4S-methylproline, and 4-methyleneproline into recombinant insulin expressed in Escherichia coli. These modified proline residues, introduced at position B28, change the biophysical properties of insulin: Incorporation of 4-methyleneproline at B28 accelerates fibril formation, while 4-methylation speeds dissociation from the pharmaceutically formulated hexamer. This work expands the scope of proline analogs amenable to incorporation into recombinant proteins and demonstrates how noncanonical amino acid mutagenesis can be used to engineer the therapeutically relevant properties of protein drugs.

A matrigel-free method for culture of pancreatic endocrine-like cells in defined protein-based hydrogels.

The transplantation of pancreatic endocrine islet cells from cadaveric donors is a promising treatment for type 1 diabetes (T1D), which is a chronic autoimmune disease that affects approximately nine million people worldwide. However, the demand for donor islets outstrips supply. This problem could be solved by differentiating stem and progenitor cells to islet cells. However, many current culture methods used to coax stem and progenitor cells to differentiate into pancreatic endocrine islet cells require Matrigel, a matrix composed of many extracellular matrix (ECM) proteins secreted from a mouse sarcoma cell line. The undefined nature of Matrigel makes it difficult to determine which factors drive stem and progenitor cell differentiation and maturation. Additionally, it is difficult to control the mechanical properties of Matrigel without altering its chemical composition. To address these shortcomings of Matrigel, we engineered defined recombinant proteins roughly 41 kDa in size, which contain cell-binding ECM peptides derived from fibronectin (ELYAVTGRGDSPASSAPIA) or laminin alpha 3 (PPFLMLLKGSTR). The engineered proteins form hydrogels through association of terminal leucine zipper domains derived from rat cartilage oligomeric matrix protein. The zipper domains flank elastin-like polypeptides whose lower critical solution temperature (LCST) behavior enables protein purification through thermal cycling. Rheological measurements show that a 2% w/v gel of the engineered proteins display material behavior comparable to a Matrigel/methylcellulose-based culture system previously reported by our group to support the growth of pancreatic ductal progenitor cells. We tested whether our protein hydrogels in 3D culture could derive endocrine and endocrine progenitor cells from dissociated pancreatic cells of young (1-week-old) mice. We found that both protein hydrogels favored growth of endocrine and endocrine progenitor cells, in contrast to Matrigel-based culture. Because the protein hydrogels described here can be further tuned with respect to mechanical and chemical properties, they provide new tools for mechanistic study of endocrine cell differentiation and maturation.

Staphylococcal secreted cytotoxins are competition sensing signals for Pseudomonas aeruginosa.

Coinfection with two notorious opportunistic pathogens, the Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus , dominates chronic pulmonary infections. While coinfection is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. Here, we dissected molecular mechanisms of interspecies sensing between P. aeruginosa and S. aureus . We discovered that P. aeruginosa senses S. aureus secreted peptides and, counterintuitively, moves towards these toxins. P. aeruginosa tolerates such a strategy through "competition sensing", whereby it preempts imminent danger/competition by arming cells with type six secretion (T6S) and iron acquisition systems. Intriguingly, while T6S is predominantly described as weaponry targeting Gram-negative and eukaryotic cells, we find that T6S is essential for full P. aeruginosa competition with S. aureus , a previously undescribed role for T6S. Importantly, competition sensing was activated during coinfection of bronchial epithelia, including T6S islands targeting human cells. This study reveals critical insight into both interspecies competition and how antagonism may cause collateral damage to the host environment.

3D-Printable Cellular Composites for the Production of Recombinant Proteins.

The incorporation of living cells into materials promises both significant challenges and new possibilities. Although recent years have seen important advances in this field, there is still much to be learned about engineering interfaces between cells and materials. Here, we present a new class of 3D-printable materials, based on poly(N-hydroxymethylacrylamide) (PNHMAA), in which the spore-forming bacterium Bacillus subtilis is effectively cross-linked into the surrounding polymeric scaffold. After dehydration and subsequent re-swelling in nutrient-rich media, embedded cells and spores become metabolically active and are capable of heterologous protein production and secretion. Strikingly, the leak-free scaffold allows protein production while preventing escape of embedded cells. The successful construction of complex three-dimensional structures by stereolithographic printing of living PNHMAA composite materials suggests utility in a broad range of applications.

Incorporation of proline analogs into recombinant proteins expressed in Escherichia coli.

Proline residues are unique in the extent to which they constrain the conformational space available to the protein backbone. Because the conformational preferences of proline cannot be recapitulated by any of the other proteinogenic amino acids, standard mutagenesis approaches that seek to introduce new chemical functionality at proline positions unavoidably perturb backbone flexibility. Here, we detail the incorporation of proline analogs into recombinant proteins in Escherichia coli via a residue-specific mutagenesis strategy. This approach results in global replacement of proline residues with high yields of the recombinant protein of interest, minimal genetic manipulation, and maintenance of backbone conformational constraints.

Genetically Programmable Microbial Assembly.

Engineered microbial communities show promise in a wide range of applications, including environmental remediation, microbiome engineering, and synthesis of fine chemicals. Here we present methods by which bacterial aggregates can be directed into several distinct architectures by inducible surface expression of heteroassociative protein domains (SpyTag/SpyCatcher and SynZip17/18). Programmed aggregation can be used to activate a quorum-sensing circuit, and aggregate size can be tuned via control of the amount of the associative protein displayed on the cell surface. We further demonstrate reversibility of SynZip-mediated assembly by addition of soluble competitor peptide. Genetically programmable bacterial assembly provides a starting point for the development of new applications of engineered microbial communities in environmental technology, agriculture, human health, and bioreactor design.

The dormancy-specific regulator, SutA, is intrinsically disordered and modulates transcription initiation in Pseudomonas aeruginosa.

Though most bacteria in nature are nutritionally limited and grow slowly, our understanding of core processes like transcription comes largely from studies in model organisms doubling rapidly. We previously identified a small protein of unknown function, SutA, in a screen of proteins synthesized in Pseudomonas aeruginosa during dormancy. SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA comprises a central α-helix with unstructured N- and C-terminal tails, and binds to the ß1 domain of RNAP. It activates transcription from the rrn promoter by both the housekeeping sigma factor holoenzyme (Eσ70 ) and the stress sigma factor holoenzyme (EσS ) in vitro, but has a greater impact on EσS . In both cases, SutA appears to affect intermediates in the open complex formation and its N-terminal tail is required for activation. The small magnitudes of in vitro effects are consistent with a role in maintaining activity required for homeostasis during dormancy. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes.

N-Myristoyl Transferase (NMT)-Catalyzed Labeling of Bacterial Proteins for Imaging in Fixed and Live Cells.

Methods for selective protein imaging are critical for elucidating how cells orchestrate fundamental biological processes. We recently developed a chemoenzymatic method to modify bacterial proteins in situ for fluorescence imaging using N-myristoyl transferase (NMT). Target proteins outfitted with an N-terminal NMT recognition sequence are covalently modified with an azido fatty acid. Subsequent strain-promoted azide-alkyne cycloaddition allows for conjugation to cell-permeant fluorophores and imaging by fluorescence microscopy. Here we describe sample preparation and labeling protocols for imaging bacterial proteins in fixed and live cells.

Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells.

Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution.

Mechanisms of Diffusion in Associative Polymer Networks: Evidence for Chain Hopping.

Networks assembled by reversible association of telechelic polymers constitute a common class of soft materials. Various mechanisms of chain migration in associative networks have been proposed; yet there remains little quantitative experimental data to discriminate among them. Proposed mechanisms for chain migration include multichain aggregate diffusion as well as single-chain mechanisms such as "walking" and "hopping", wherein diffusion is achieved by either partial ("walking") or complete ("hopping") disengagement of the associated chain segments. Here, we provide evidence that hopping can dominate the effective diffusion of chains in associative networks due to a strong entropic penalty for bridge formation imposed by local network structure; chains become conformationally restricted upon association with two or more spatially separated binding sites. This restriction decreases the effective binding strength of chains with multiple associative domains, thereby increasing the probability that a chain will hop. For telechelic chains this manifests as binding asymmetry, wherein the first association is effectively stronger than the second. We derive a simple thermodynamic model that predicts the fraction of chains that are free to hop as a function of tunable molecular and network properties. A large set of self-diffusivity measurements on a series of model associative polymers finds good agreement with this model.

Incorporation of Non-Canonical Amino Acids into Proteins by Global Reassignment of Sense Codons.

Non-canonical amino acids are finding increasing use in basic and applied research. Proteins that evolved naturally for biological function did so by exploiting the chemistries of the canonical amino acids; however, when proteins are repurposed for biomedical and pharmacological applications, they are often subject to conditions different from those characteristic of their original biological environments. Non-canonical amino acids can impart properties that are inaccessible within canonical protein sequence space, and can thereby lead to improved or new functionality. We describe simple methods for global replacement of canonical amino acids by their non-canonical counterparts in recombinant proteins made in high yield in bacterial expression hosts. These methods can be used to engineer both chemical and physical properties of recombinant proteins.

Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro.

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Cell-type-specific metabolic labeling of nascent proteomes in vivo.

Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.

Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms.

Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.

Protein-Mediated Colloidal Assembly.

Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein-protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein-protein interactions can be used to assemble complex core-shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation.

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of noncanonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.

A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome-mRNA Interactions in Single Cells.

Single-molecule fluorescence in situ hybridization (smFISH) is a simple and widely used method to measure mRNA transcript abundance and localization in single cells. A comparable single-molecule in situ method to measure mRNA translation would enable a more complete understanding of gene regulation. Here we describe a fluorescence assay to detect ribosome interactions with mRNA (FLARIM). The method adapts smFISH to visualize and characterize translation of single molecules of mRNA in fixed cells. To visualize ribosome-mRNA interactions, we use pairs of oligonucleotide probes that bind separately to ribosomes (via rRNA) and to the mRNA of interest, and that produce strong fluorescence signals via the hybridization chain reaction (HCR) when the probes are in close proximity. FLARIM does not require genetic manipulation, is applicable to practically any endogenous mRNA transcript, and provides both spatial and temporal information. We demonstrate that FLARIM is sensitive to changes in ribosome association with mRNA upon inhibition of global translation with puromycin. We also show that FLARIM detects changes in ribosome association with an mRNA whose translation is upregulated in response to increased concentrations of iron.