Archaeal Connectase is a specific and efficient protein ligase related to proteasome ß subunits.

Sequence-specific protein ligations are widely used to produce customized proteins "on demand." Such chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins are generated using a range of chemical, (split) intein, split domain, or enzymatic methods. Where short ligation motifs and good chemoselectivity are required, ligase enzymes are often chosen, although they have a number of disadvantages, for example poor catalytic efficiency, low substrate specificity, and side reactions. Here, we describe a sequence-specific protein ligase with more favorable characteristics. This ligase, Connectase, is a monomeric homolog of 20S proteasome subunits in methanogenic archaea. In pulldown experiments with Methanosarcina mazei cell extract, we identify a physiological substrate in methyltransferase A (MtrA), a key enzyme of archaeal methanogenesis. Using microscale thermophoresis and X-ray crystallography, we show that only a short sequence of about 20 residues derived from MtrA and containing a highly conserved KDPGA motif is required for this high-affinity interaction. Finally, in quantitative activity assays, we demonstrate that this recognition tag can be repurposed to allow the ligation of two unrelated proteins. Connectase catalyzes such ligations at substantially higher rates, with higher yields, but without detectable side reactions when compared with a reference enzyme. It thus presents an attractive tool for the development of new methods, for example in the preparation of selectively labeled proteins for NMR, the covalent and geometrically defined attachment of proteins on surfaces for cryo-electron microscopy, or the generation of multispecific antibodies.

Water Harvesting at the Single-Crystal Level.

Metal-organic frameworks (MOFs) have emerged as a class of porous materials with facile uptake and release of water, turning them into excellent substrates for real-world atmospheric water harvesting applications. The performance of different MOF systems was experimentally characterized at the bulk level by assessing the total amount of water taken up and the release kinetics, leaving the question behind of what the upper limit of the pristine materials actually is. Moreover, recent devices rely on fluidized bed reactors that exploit the harvesting capacities of MOFs at the single-crystal (SC) level. In this publication, we present a novel methodology based on Raman spectroscopy, for acquiring water adsorption isotherms and kinetic curves with a sub-micrometer resolution that provides valuable insights into the material behavior probing the pristine MOF at the SC level. We investigated isolated MOF-801 particles in situ and could dissect contributions of intra- and inter-particle effects on the water harvesting performance of MOF-801 via adsorption-desorption isotherms and kinetic curves. Using spontaneous Raman spectroscopy, we found an almost 20-fold faster uptake for the undisturbed crystalline material. Correlative imaging based on four-wave mixing and coherent anti-Stokes Raman scattering further localized the uptaken water inside MOF-801 and identified inter-particle condensation as the main source for the discrepancies between the performance at the bulk and SC level. Our studies determined an upper limit of around 91.9 L/kgMOF/day for MOF-801.

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides cth GAMP.

The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2'3'-cth GAMP and 3'3'-cth GAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2'3'-cth GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-cth GAMP in THP-1 cells.

An astonishing wealth of new proteasome homologs.

MOTIVATION: The proteasome is the main proteolytic machine for targeted protein degradation in archaea and eukaryotes. While some bacteria also possess the proteasome, most of them contain a simpler and more specialized homolog, the heat shock locus V protease. In recent years, three further homologs of the proteasome core subunits have been characterized in prokaryotes: Anbu, BPH and connectase. With the inclusion of these members, the family of proteasome-like proteins now exhibits a range of architectural and functional forms, from the canonical proteasome, a barrel-shaped protease without pronounced intrinsic substrate specificity, to the monomeric connectase, a highly specific protein ligase. RESULTS: We employed systematic sequence searches to show that we have only seen the tip of the iceberg so far and that beyond the hitherto known proteasome homologs lies a wealth of distantly related, uncharacterized homologs. We describe a total of 22 novel proteasome homologs in bacteria and archaea. Using sequence and structure analysis, we analyze their evolutionary history and assess structural differences that may modulate their function. With this initial description, we aim to stimulate the experimental investigation of these novel proteasome-like family members. AVAILABILITY AND IMPLEMENTATION: The protein sequences in this study are searchable in the MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) with ProtBLAST/PSI-BLAST and with HHpred (database 'proteasome_homologs'). The following data are available at https://data.mendeley.com/datasets/t48yhff7hs/3: (i) sequence alignments for each proteasome-like homolog, (ii) the coordinates for their structural models and (iii) a cluster-map file, which can be navigated interactively in CLANS and gives direct access to all the sequences in this study. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

On the Origins of Symmetry and Modularity in the Proteasome Family: Symmetry Transitions are Pivotal in the Evolution and Functional Diversification of Self-Compartmentalizing Proteases.

The proteasome family of proteases comprises oligomeric assemblies of very different symmetry. In different sizes, it features ring-like oligomers with dihedral symmetry that allow the stacking of further rings of regulatory subunits as observed in the modular proteasome system, but also less symmetric helical assemblies. Comprehensive sequence and structural analyses of proteasome homologs reveal a parsimonious scenario of how symmetry may have emerged from a monomeric ancestral precursor and how it may have evolved throughout the proteasome family. The four characterized representatives-ancestral ß subunit (Anbu), HslV, betaproteobacterial proteasome homolog (BPH), and the 20S proteasome-are outlasting cornerstones in the family's evolutionary history, each marking a transition in symmetry. This article contextualizes the evolutionary and functional key aspects of these symmetry transitions, explaining how they facilitated the diversification and concurrent evolution of independent proteolytic systems side by side, each with its customized network of auxiliary interactors.

Effect of Chain-End Chemistries on the Efficiency of Coupling Antibodies to Polymers Using Unnatural Amino Acids.

Novel conjugates that incorporate strategies for increasing the therapeutic payload, such as targeted polymeric delivery vehicles, have great potential in overcoming limitations of conventional antibody therapies that often exhibit immunogenicity and limited drug loading. Click chemistry has significantly expanded the toolbox of effective strategies for developing hybrid polymer-biomolecule conjugates, however, effective systems require orthogonality between the polymer and biomolecule chemistries to achieve efficient coupling. Here, three cycloaddition-based strategies for antibody conjugation to polymeric carriers are explored and show that a purely radical-based method for polymer synthesis and subsequent biomolecule attachment has a trade-off between coupling efficiency of the antibody and the ability to synthesize polymers with controlled chemical properties. It is shown that careful consideration of both coupling chemistries as well as the potential effect of how this modulates the chemical properties of the polymer nanocarrier should be considered during the development of such systems. The strategies described offer insight into improving conjugate development for therapeutic and theranostic applications. In this system, polymerization using conventional and established reversible addition fragmentation chain transfer (RAFT) agents, followed by multiple post-modification steps, always leads to systems with more defined chemical architectures compared to strategies that utilize alkyne-functional RAFT agents.

Structural characterization of the bacterial proteasome homolog BPH reveals a tetradecameric double-ring complex with unique inner cavity properties.

Eukaryotic and archaeal proteasomes are paradigms for self-compartmentalizing proteases. To a large extent, their function requires interplay with hexameric ATPases associated with diverse cellular activities (AAA+) that act as substrate unfoldases. Bacteria have various types of self-compartmentalizing proteases; in addition to the proteasome itself, these include the proteasome homolog HslV, which functions together with the AAA+ HslU; the ClpP protease with its partner AAA+ ClpX; and Anbu, a recently characterized ancestral proteasome variant. Previous bioinformatic analysis has revealed a novel bacterial member of the proteasome family Betaproteobacteria proteasome homolog (BPH). Using cluster analysis, we here affirmed that BPH evolutionarily descends from HslV. Crystal structures of the Thiobacillus denitrificans and Cupriavidus metallidurans BPHs disclosed a homo-oligomeric double-ring architecture in which the active sites face the interior of the cylinder. Using small-angle X-ray scattering (SAXS) and electron microscopy averaging, we found that BPH forms tetradecamers in solution, unlike the dodecamers seen in HslV. Although the highly acidic inner surface of BPH was in striking contrast to the cavity characteristics of the proteasome and HslV, a classical proteasomal reaction mechanism could be inferred from the covalent binding of the proteasome-specific inhibitor epoxomicin to BPH. A ligand-bound structure implied that the elongated BPH inner pore loop may be involved in substrate recognition. The apparent lack of a partner unfoldase and other unique features, such as Ser replacing Thr as the catalytic residue in certain BPH subfamilies, suggest a proteolytic function for BPH distinct from those of known bacterial self-compartmentalizing proteases.

Effects of Surface Charge of Hyperbranched Polymers on Cytotoxicity, Dynamic Cellular Uptake and Localization, Hemotoxicity, and Pharmacokinetics in Mice.

Nanoscaled polymeric materials are increasingly being investigated as pharmaceutical products, drug/gene delivery vectors, or health-monitoring devices. Surface charge is one of the dominant parameters that regulates nanomaterial behavior in vivo. In this paper, we demonstrated how control over chemical synthesis allowed manipulation of nanoparticle surface charge, which in turn greatly influenced the in vivo behavior. Three methacrylate/methacrylamide-based monomers were used to synthesize well-defined hyperbranched polymers (HBP) by reversible addition-fragmentation chain transfer (RAFT) polymerization. Each HBP had a hydrodynamic diameter of approximately 5 nm as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Incorporation of a fluorescent moiety within the polymeric nanoparticles allowed determination of how charge affected the in vivo pharmacokinetic behavior of the nanomaterials and the biological response to them. A direct correlation between surface charge, cellular uptake, and cytotoxicity was observed, with cationic HBPs exhibiting higher cellular uptake and cytotoxicity than their neutral and anionic counterparts. Evaluation of the distribution of the differently charged HBPs within macrophages showed that all HBPs accumulated in the cytoplasm, but cationic HBPs also trafficked to, and accumulated within, the nucleus. Although cationic HBPs caused slight hemolysis, this was generally below accepted levels for in vivo safety. Analysis of pharmacokinetic behavior showed that cationic and anionic HBPs had short blood half-lives of 1.82 ± 0.51 and 2.34 ± 0.93 h respectively, compared with 5.99 ± 2.30 h for neutral HBPs. This was attributed to the fact that positively charged surfaces are more readily covered with opsonin proteins and thus more visible to phagocytic cells. This was supported by in vitro flow cytometric and qualitative live cell imaging studies, which showed that cationic HBPs tended to be taken up by macrophages more effectively and rapidly than neutral and anionic particles.

Using Peptide Aptamer Targeted Polymers as a Model Nanomedicine for Investigating Drug Distribution in Cancer Nanotheranostics.

Theranostics is a strategy that combines multiple functions such as targeting, stimulus-responsive drug release, and diagnostic imaging into a single platform, often with the aim of developing personalized medicine.1,2 Based on this concept, several well-established hyperbranched polymeric theranostic nanoparticles were synthesized and characterized as model nanomedicines to investigate how their properties affect the distribution of loaded drugs at both the cell and whole animal levels. An 8-mer peptide aptamer was covalently bound to the periphery of the nanoparticles to achieve both targeting and potential chemosensitization functionality against heat shock protein 70 (Hsp70). Doxorubicin was also bound to the polymeric carrier as a model chemotherapeutic drug through a degradable hydrazone bond, enabling pH-controlled release under the mildly acid conditions that are found in the intracellular compartments of tumor cells. In order to track the nanoparticles, cyanine-5 (Cy5) was incorporated into the polymer as an optical imaging agent. In vitro cellular uptake was assessed for the hyperbranched polymer containing both doxorubicin (DOX) and Hsp70 targeted peptide aptamer in live MDA-MB-468 cells, and was found to be greater than that of either the untargeted, DOX-loaded polymer or polymer alone due to the specific affinity of the peptide aptamer for the breast cancer cells. This was also validated in vivo with the targeted polymers showing much higher accumulation within the tumor 48 h postinjection than the untargeted analogue. More detailed assessment of the nanomedicine distribution was achieved by directly following the polymeric carrier and the doxorubicin at both the in vitro cellular level via compartmental analysis of confocal images of live cells and in whole tumors ex vivo using confocal imaging to visualize the distribution of the drug in tumor tissue as a function of distance from blood vessels. Our results indicate that this polymeric carrier shows promise as a cancer theranostic, demonstrating active targeting to tumor cells with the capability for simultaneous drug release.

Targeting Nanomedicines to Prostate Cancer: Evaluation of Specificity of Ligands to Two Different Receptors In Vivo.

PURPOSE: This manuscript utilised in vivo multispectral imaging to demonstrate the efficacy of two different nanomedicine formulations for targeting prostate cancer. METHODS: Pegylated hyperbranched polymers were labelled with fluorescent markers and targeting ligands against two different prostate cancer markers; prostate specific membrane antigen (PSMA) and the protein kinase, EphrinA2 receptor (EphA2). The PSMA targeted nanomedicine utilised a small molecule glutamate urea inhibitor of the protein, while the EphA2 targeted nanomedicine was conjugated to a single-chain variable fragment based on the antibody 4B3 that has shown high affinity to the receptor. RESULTS: Hyperbranched polymers were synthesised bearing the different targeting ligands. In the case of the EphA2-targeting nanomedicine, significant in vitro uptake was observed in PC3 prostate cancer cells that overexpress the receptor, while low uptake was observed in LNCaP cells (that have minimal expression of this receptor). Conversely, the PSMA-targeted nanomedicine showed high uptake in LNCaP cells, with only minor uptake in the PC3 cells. In a dual-tumour xenograft mouse model, the nanomedicines showed high uptake in tumours in which the receptor was overexpressed, with only minimal non-specific accumulation in the low-expression tumours. CONCLUSIONS: This work highlighted the importance of clearly defining the target of interest in next-generation nanomedicines, and suggests that dual-targeting in such nanomedicines may be a means to achieve greater efficacy.

Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

Sticky water surfaces: helix-coil transitions suppressed in a cell-penetrating peptide at the air-water interface.

GALA is a 30 amino acid synthetic peptide consisting of a Glu-Ala-Leu-Ala repeat and is known to undergo a reversible structural transition from a disordered to an α-helical structure when changing the pH from basic to acidic values. In its helical state GALA can insert into and disintegrate lipid membranes. This effect has generated much interest in GALA as a candidate for pH triggered, targeted drug delivery. GALA also serves as a well-defined model system to understand cell penetration mechanisms and protein folding triggered by external stimuli. Structural transitions of GALA in solution have been studied extensively. However, cell penetration is an interfacial effect and potential biomedical applications of GALA would involve a variety of surfaces, e.g., nanoparticles, lipid membranes, tubing, and liquid-gas interfaces. Despite the apparent importance of interfaces in the functioning of GALA, the effect of surfaces on the reversible folding of GALA has not yet been studied. Here, we use sum frequency generation vibrational spectroscopy (SFG) to probe the structural response of GALA at the air-water interface and IR spectroscopy to follow GALA folding in bulk solution. We combine the SFG data with molecular dynamics simulations to obtain a molecular-level picture of the interaction of GALA with the air-water interface. Surprisingly, while the fully reversible structural transition was observed in solution, at the water-air interface, a large fraction of the GALA population remained helical at high pH. This "stickiness" of the air-water interface can be explained by the stabilizing interactions of hydrophobic leucine and alanine side chains with the water surface.

Detection and quantification of C-terminally tagged proteins by in-gel fluorescence.

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.

Specific, sensitive and quantitative protein detection by in-gel fluorescence.

Recombinant proteins in complex solutions are typically detected with tag-specific antibodies in Western blots. Here we describe an antibody-free alternative in which tagged proteins are detected directly in polyacrylamide gels. For this, the highly specific protein ligase Connectase is used to selectively fuse fluorophores to target proteins carrying a recognition sequence, the CnTag. Compared to Western blots, this procedure is faster, more sensitive, offers a better signal-to-noise ratio, requires no optimization for different samples, allows more reproducible and accurate quantifications, and uses freely available reagents. With these advantages, this method represents a promising alternative to the state of the art and may facilitate studies on recombinant proteins.

Biomimetic silver-containing colloids of poly(2-methacryloyloxyethyl phosphorylcholine) and their film-formation properties.

The synthesis of stable dispersions of hybrid colloids comprising copolymers of biocompatible 2-hydroxyethyl methacrylate (HEMA) and zwitterionic, biomimetic 2-methacryloyloxyethyl phosphorylcholine (MPC) incorporating antibacterial AgBF(4) by inverse miniemulsion is described. The prepared hybrid colloids were designed to provide both antibacterial and antifouling properties for the formation of interesting, multifunctional films. The obtained particles had sizes in the range of 130-160 nm with two different weight ratios of MPC to HEMA (1:10 and 2:5) and AgBF(4) contents between 0% and 15%. The silver salt takes on the role of the lipophobe in stabilizing the miniemulsion droplets against Ostwald ripening and is reduced after polymerization to Ag nanoparticles by gaseous hydrazine. Subsequently, the hybrid particles are transformed into smooth and stable films with thicknesses between 145 and 225 nm by simple drop casting and solvent annealing. The dispersions and films were thoroughly characterized by DLS, TEM, SEM, EDX, TGA, UV-vis spectroscopy, ICP-OES, XRD, AFM, and contact angle measurements. After immersion into water, the films did not show detectable leakage of silver, so they could be employed as dual-functional antifouling and antibacterial coatings.

Single Crystals Heterogeneity Impacts the Intrinsic and Extrinsic Properties of Metal-Organic Frameworks.

At present, an enormous characterization gap exists between the study of the crystal structure of a material and its bulk properties. Individual particles falling within this gap cannot be fully characterized in a correlative manner by current methods. The authors address this problem by exploiting the noninvasive nature of optical microscopy and spectroscopy for the correlative analysis of metal-organic framework particles in situ. They probe the intrinsic as well as extrinsic properties in a correlated manner. The authors show that the crystal shape of MIL-88A strongly impacts its optical absorption. Furthermore, the question of how homogeneously water is distributed and adsorbed within one of the most promising materials for harvesting water from humid air, MOF-801, is addressed. The results demonstrate the considerable importance of the particle level and how it can affect the property of the material.

Spatio-selective activation of nuclear translocation of YAP with light directs invasion of cancer cell spheroids.

The mechanical properties of the extracellular matrix strongly influence tumor progression and invasion. Yes-associated protein (YAP) has been shown to be a key regulator of this process translating mechanical cues from the extracellular matrix into intracellular signals. Despite its apparent role in tumor progression and metastasis, it is not clear yet, whether YAP activation can actively trigger the onset of invasion. To address this question, we designed a photo-activatable YAP (optoYAP), which allows for spatiotemporal control of its activation. The activation mechanism of optoYAP is based on optically triggered nuclear translocation of the protein. Activation of optoYAP induces downstream signaling for several hours and leads to increased proliferation in two- and three-dimensional cultures. Applied to cancer spheroids, optoYAP activation induces invasion. Site-selective activation of optoYAP in cancer spheroids strikingly directs invasion into the activated direction. Thus, nuclear translocation of YAP may be enough to trigger the onset of invasion.

Sponges as bioindicators for microparticulate pollutants?

Amongst other threats, the world's oceans are faced with man-made pollution, including an increasing number of microparticulate pollutants. Sponges, aquatic filter-feeding animals, are able to incorporate fine foreign particles, and thus may be a potential bioindicator for microparticulate pollutants. To address this question, 15 coral reef demosponges sampled around Bangka Island (North Sulawesi, Indonesia) were analyzed for the nature of their foreign particle content using traditional histological methods, advanced light microscopy, and Raman spectroscopy. Sampled sponges accumulated and embedded the very fine sediment fraction (<200 µm), absent in the surrounding sand, in the ectosome (outer epithelia) and spongin fibers (skeletal elements), which was confirmed by two-photon microscopy. A total of 34 different particle types were identified, of which degraded man-made products, i.e., polystyrene, particulate cotton, titanium dioxide and blue-pigmented particles, were incorporated by eight specimens at concentrations between 91 and 612 particle/g dry sponge tissue. As sponges can weigh several hundreds of grams, we conservatively extrapolate that sponges can incorporate on average 10,000 microparticulate pollutants in their tissue. The uptake of particles, however, appears independent of the material, which suggests that the fluctuation in material ratios is due to the spatial variation of surrounding microparticles. Therefore, particle-bearing sponges have a strong potential to biomonitor microparticulate pollutants, such as microplastics and other degraded industrial products.

Targeted and modular architectural polymers employing bioorthogonal chemistry for quantitative therapeutic delivery.

There remain several key challenges to existing therapeutic systems for cancer therapy, such as quantitatively determining the true, tissue-specific drug release profile in vivo, as well as reducing side-effects for an increased standard of care. Hence, it is crucial to engineer new materials that allow for a better understanding of the in vivo pharmacokinetic/pharmacodynamic behaviours of therapeutics. We have expanded on recent "click-to-release" bioorthogonal pro-drug activation of antibody-drug conjugates (ADCs) to develop a modular and controlled theranostic system for quantitatively assessing site-specific drug activation and deposition from a nanocarrier molecule, by employing defined chemistries. The exploitation of quantitative imaging using positron emission tomography (PET) together with pre-targeted bioorthogonal chemistries in our system provided an effective means to assess in real-time the exact amount of active drug administered at precise sites in the animal; our methodology introduces flexibility in both the targeting and therapeutic components that is specific to nanomedicines and offers unique advantages over other technologies. In this approach, the in vivo click reaction facilitates pro-drug activation as well as provides a quantitative means to investigate the dynamic behaviour of the therapeutic agent.