Small Molecule Substrates Research Articles

Dynamic mapping of proteome trafficking within and between living cells by TransitID

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

A general model to predict small molecule substrates of enzymes based on machine and deep learning

For most proteins annotated as enzymes, it is unknown which primary and/or secondary reactions they catalyze. Experimental characterizations of potential substrates are time-consuming and costly. Machine learning predictions could provide an efficient alternative, but are hampered by a lack of information regarding enzyme non-substrates, as available training data comprises mainly positive examples. Here, we present ESP, a general machine-learning model for the prediction of enzyme-substrate pairs with an accuracy of over 91% on independent and diverse test data. ESP can be applied successfully across widely different enzymes and a broad range of metabolites included in the training data, outperforming models designed for individual, well-studied enzyme families. ESP represents enzymes through a modified transformer model, and is trained on data augmented with randomly sampled small molecules assigned as non-substrates. By facilitating easy in silico testing of potential substrates, the ESP web server may support both basic and applied science.

Structural insight into TIPE1 functioning as a lipid transfer protein

As a member of the tumor necrosis factor-α-induced protein 8 (TNFAIP8/TIPE) family, TIPE1 has been found to be associated with many cellular signaling pathways in regulating apoptosis, autophagy, and tumorigenesis. However, the position of TIPE1 in the signaling network remains elusive. Here we present the crystal structure of zebrafish TIPE1 in complex with phosphatidylethanolamine (PE) at a resolution of 1.38 Å. By comparison with structures of other three TIPE family proteins, a universal phospholipid-binding mode was proposed. Namely, the hydrophobic cavity binds to fatty acid tails, while ‘X-R-R’ triad nearby the entrance of cavity recognizes the phosphate group head. Using molecular dynamics (MD) simulations, we further elaborated the mechanism of how the lysine-rich N-terminal domain assisting TIPE1 to favorably bind to phosphatidylinositol (PI). Beside small molecule substrate, we identified Gαi3 as a direct-binding partner of TIPE1 using GST pull-down assay and size-exclusion chromatography. Analyses of key-residue mutations and predicted complex structure revealed that the binding mode of TIPE1 to Gαi3 could be non-canonical. In summary, our findings narrowed down TIPE1’s position in Gαi3-related and PI-inducing signaling pathways.Communicated by Ramaswamy H. Sarma

An improved lysozyme substrate preparation method for the biochemistry kinetics laboratory

The mutation and growth of Bacteriophage T4 Lysozyme (T4L) is easily accomplished in an undergraduate biochemistry laboratory and allows students to visualize how mutations affect enzyme stability and kinetics. However, full Michaelis-Menten parameters have been difficult to observe due to lack of a readily available, small molecule substrate. A FITC-tagged peptidoglycan has been used previously, but it is not commercially available and its preparation method was not appropriate for the undergraduate laboratory. Here, we describe a cost-effective, non-toxic, and reproducible means of generating FITC-tagged peptidoglycan fragments. Fluorescence assays monitoring substrate digestion using HEWL and T4L produced signals sufficient for reliable analysis of lysozyme kinetics.

Using solid-supported membrane electrophysiology to probe the transport mechanism of EmrE.

Multi-drug-resistant bacterial infections pose a major threat to public health. As case numbers continue to rise and new strains are discovered with resistance to existing antibiotics, it is increasingly imperative to develop novel treatments. The recent discovery that the model multi-drug efflux pump from Escherichia coli, EmrE, can perform multiple types of transport suggests this may be a compelling target for therapeutic intervention. Initial studies have identified several small-molecule substrates capable of inducing transporter-dependent susceptibility rather than the well-known antibiotic resistance phenotype. However, many questions regarding the underlying mechanism and regulation of this transporter still remain. Here we use solid-supported membrane electrophysiology to refine our mechanistic understanding of EmrE transport by systematically examining the regulation of transporter activity. Using small molecule substrates with diverse chemical properties in tandem with protein mutagenesis we examine the role of substrate interactions in modulating the net transport through EmrE. Careful manipulation of substrate concentrations and gradient magnitudes allows for examination of net flux under different conditions while repeated washings allow for exchange of internal buffer conditions to enable high-throughput analysis. With a high degree of familial conservation in addition to a suggested role in transporter evolution, mechanistic insight into the transport regulation of EmrE may be broadly applicable across multi-drug resistant efflux pumps. By examining the innate modalities for regulation within the protein and how the molecular features of various substrates interact with these entitles we add to the growing body of knowledge supporting this system as a tractable target for novel antibiotic development.

Structural basis for the recruitment of PARP4 and NAD+to the vault complex.

Vault is a massive ribonucleoprotein complex that contains 78 copies of the major vault protein (MVP), which oligomerize into a barrel-like cage. This cage envelops several constitutively-bound minor vault components as well as transiently interacting “cargo” molecules. Vault mediates key biological functions including cell metabolism, innate immunity, and the cell's responses to disparate stressors. Previous studies suggest that vault may contribute to these processes by regulating cell signaling or guiding the intracellular localization of potential cargo, but its molecular-level mechanism remains unclear. In this study, we shed light on vault's functional mechanism by using cryo-EM to resolve the structure of human MVP in complex with the understudied minor vault component poly (ADP)-ribose polymerase 4 (PARP4) and PARP4's small-molecule substrate, nicotinamide adenine dinucleotide (NAD+). PARP4 is an ADP-ribosyltransferase that relies on available NAD+ to deposit ADP-ribose moieties onto macromolecular targets. It therefore has the potential to direct vault-mediated signaling activity, modify its cargo, or alter the vault cage architecture. Our sub-3.5 Å structures are the first to characterize MVP and PARP4 binding at the atomic level, and they also reveal a surprising interaction between NAD+ and a highly conserved region of MVP. Intriguingly, the presence of NAD+ causes the N-terminal region of PARP4 to undergo a substantial conformational change within the vault cage, drawing it into proximity with MVP's NAD+ binding pocket. Experiments are underway to determine the strength of the interaction between MVP and NAD+, MVP's specificity for the metabolite versus other nucleotides, and the extent to which the interaction between MVP and NAD+ impacts PARP4 activity.

Catalytic length-controlled oligomerization with synthetic programmable templates

Nature uses templated length-controlled oligomerization to process genetic information. Templates that are DNA and RNA based and fully synthetic have also been developed for preparing unnatural oligomers. However, these reactions require stoichiometric amounts of the template for product formation. Here we report a catalytic macrocyclic template that promotes the oligomerization of a small-molecule substrate with a remarkable degree of length control. The design of the template is based on rigid oligoproline moieties decorated with catalytic sites in a defined spatial arrangement. The dimension of the macrocycle and the number of catalytic moieties determine the number of monomers that are incorporated into the growing oligomer, thus allowing access to specific products with lengths preprogrammed by the template.

Proteomics analysis reveals that CirA in Aeromonas hydrophila is involved in nutrient uptake.

The colicin I receptor (CirA) is a well-studied outer membrane protein that has been reported to play important roles in antibiotic resistance, virulence, and iron homeostasis, although its exact physiological roles require further investigation. In this study, differentially expressed proteins between the ΔahcirA and wild-type (WT) strains of Aeromonas hydrophila were compared using quantitative proteomics. Bioinformatics analysis revealed that the expression of peptide, histidine, and arginine ATP-binding cassette (ABC) transporter system-related proteins was significantly higher in the ΔahcirA strain. Subsequent growth assays revealed that ΔahcirA grew slower than the WT strain in nutrient-limited medium when supplemented with dipeptide, histidine, and arginine as the carbon source. Far-western blot analysis further confirmed that AhCirA can directly bind to histidine/arginine and dipeptide small-molecule substrates in addition to their periplasmic-binding proteins, AhDppA and AhHisJ, respectively. These results indicate that AhCirA may play an important role in the uptake of amino acids and peptides as a channel-forming porin while also directly interacting with ABC transporters to transport nutrient substances into the plasma membrane. Overall, this study demonstrates that AhCirA is a multifunctional protein in A. hydrophila and extends our understanding of known nutrient transport mechanisms among bacteria.

Biosynthesis of Isonitrile Lipopeptide Metallophores from Pathogenic Mycobacteria.

Isonitrile lipopeptides (INLPs) are known to be related to the virulence of pathogenic mycobacteria by mediating metal transport, but their biosynthesis remains obscure. In this work, we use in vitro biochemical assays, site-directed mutagenesis, chemical synthesis, and spectroscopy techniques to scrutinize the activity of core enzymes required for INLP biosynthesis in mycobacteria. Compared to environmental Streptomyces, pathogenic Mycobacterium employ a similar chemical logic and enzymatic machinery in INLP biosynthesis, differing mainly in the fatty-acyl chain length, which is controlled by multiple enzymes in the pathway. Our in-depth study on the non-heme iron(II) and α-ketoglutarate-dependent dioxygenase for isonitrile generation, including Rv0097 from Mycobacterium tuberculosis (Mtb), demonstrates that it recognizes a free-standing small molecule substrate, different from the recent hypothesis that a carrier protein is required for Rv0097 in Mtb. A key residue in Rv0097 is further identified to dictate the varied fatty-acyl chain length specificity between Streptomyces and Mycobacterium.

Catalytic templated length-controlled oligomerization.

Templated synthesis is an intriguing strategy for the length-controlled synthesis of oligomers. Traditionally, such reactions require stoichiometric amounts of the template with respect to the product. Recently we reported catalytic macrocyclic templates that promote oligomerization of a small molecule substrate with a remarkable degree of length control. Herein we present our efforts toward creating linear templates for catalytic length-controlled oligomer synthesis.

Assembly of split aptamers by dynamic pH-responsive covalent ligation.

Reversible pH-responsive N-methoxyoxazolidine formation is used to ligate split aptamer fragments. Two twice-split models and one thrice-split model of CBA (cocaine-binding aptamer) were examined. The aptamer assembly was dynamic, proportional to the substrate concentration and occurred without interfering background ligation.

Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility.

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

A chemical approach for the specific generation of cysteine sulfinylation

Oxidative modifications of protein cysteines play a crucial role in regulating redox homeostasis, redox signaling, and protein function. One of the modifications involves the oxidation of a cysteine thiol to a sulfinic acid (R-SO2H) which has been considered a marker for oxidative stress and an important activator for certain proteins. Here we report a protocol for the selective formation of sulfinic acids on cysteine residues in a simple 3-step operation under mild conditions: thiol-blocking, oxidation, and reduction. The specificity and efficiency of the method were demonstrated by using both small molecule substrates and a protein model.

Optimized Data Set and Feature Construction for Substrate Prediction of Membrane Transporters.

α-Helical transmembrane proteins termed membrane transporters mediate the passage of small hydrophilic substrate molecules across biological lipid bilayer membranes. Annotating the specific substrates of the dozens to hundreds of individual transporters of an organism is an important task. In the past, machine learning classifiers have been successfully trained on pan-organism data sets to predict putative substrates of transporters. Here, we critically examine the selection of an optimal data set of protein sequence features for the classification task. We focus on membrane transporters of the three model organisms Escherichia coli, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as human. We show that organism-specific classifiers can be robustly trained if at least 20 samples are available for each substrate class. If information from position-specific scoring matrices is included, such classifiers have F1 scores between 0.85 and 1.00. For the largest data set (A. thaliana), a 4-class classifier yielded an F-score of 0.97. On a pan-organism data set composed of transporters of all four organisms, amino acid and sugar transporters were predicted with an F1 score of 0.91.

A Novel Substrate for Assessing Thrombin Activity in Whole Blood

At sites of vessel injury, thrombin acts as the central mediator of coagulation through catalyzing fibrin clot formation and platelet activation. Currently, thrombin generation is measured using small molecule substrates. These substrates are limited in that they detect both active thrombin and thrombin bound to its inhibitor α2-macroglobulin (α2-M) and have limited utility in whole blood (Ninivaggi et al. Clin Chem 2012). In addition, these substrates contain short thrombin recognition sequences ranging from 3-4 amino acids and are unable to form additional exosite interactions with thrombin, which limit their specificity (Gallwitz et al. Plos One 2012). Furthermore, they are unfeasible for in vivo use because their hydrophobicity allows them to enter cells. However, plasma assays are limited because they ignore the hemostatic contributions of red blood cells, platelets and leukocytes and require anticoagulation and the addition of exogenous coagulation activators (Swystun & Liaw. Blood 2016 & do Amaral et al. Stem Cells Int 2016). Thus, we have developed a fluorescence resonance energy transfer (FRET)-based thrombin sensor to overcome these limitations. We cloned and expressed the construct encoding the FRET-based thrombin substrate in Escherichia coli and purified the substrate using affinity chromatography. We confirmed using Sodium dodecyl sulfate−polyacrylamide gel electrophoresis that the substrate was successfully purified and that thrombin cleavage of the purified substrate changes its FRET efficiency by monitoring its spectrum of emission. To determine whether the FRET-based thrombin substrate can form interactions with thrombin's anion binding exosites and assess its potential for in vivo use, we measured the kinetics parameters of the substrate for thrombin in the presence of exosite inhibitors and thrombin's exosite variants as well as mouse thrombin and plasmin. We then compared the resulting kinetics parameters with commercially available thrombin substrates S2238 and technothrombin. We show that unlike S2238 and technothrombin, our FRET-based thrombin sensor forms strong interactions with thrombin's anion binding exosite I and displays preference for free thrombin compared to thrombomodulin (TM) bound thrombin which implied its preference for procoagulant thrombin rather than anticoagulant TM bound thrombin that activates protein C. Furthermore, our FRET-based thrombin substrate has potential to be used in mouse models because it can be efficiently cleaved by mouse thrombin and is relatively plasmin resistant. To assess the specificity of the FRET-based thrombin substrate for thrombin, we compared the rate of hydrolysis of the substrate by thrombin and several other enzymes in circulation and compared the results to S2238 and technothrombin. Our results indicated that when compared to commercially available thrombin substrates, our FRET-based thrombin substrate is highly specific for thrombin. Since the thrombin inhibitor α2-M is present in circulation, we also compared the rate of hydrolysis of the 3 substrates for α2-M bound thrombin and free thrombin. Unlike S2238 and technothrombin, our FRET-based thrombin substrate is uncleaved by α2-M bound thrombin. The key properties of the FRET-based thrombin substrate in comparison to S22238 and technothrombin are summarized in Table 1. We then measured thrombin generation in plasma using our FRET-based thrombin substrate and showed that it is sensitive to direct oral anticoagulants in ways that are consistent with literature findings where thrombin generation was measured using technothrombin (Sivaraja et al. Plos One 2018 & Douxfils et al. Thromb Res 2012). We also confirmed that the FRET-based thrombin substrate itself is not a coagulation inhibitor since its presence did not alter thrombin generation parameters measured using technothrombin. Lastly, we confirmed that the FRET-based thrombin substrate can measure thrombin generation in whole blood since it successfully produced thrombin generation curves. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chromophore Nanohybrids for Sensing and Singlet Oxygen Generation

Nanohybrid materials can exhibit the physical properties of their components and be used in various applications. Also, novel chromophores involving synthetically flexible molecules such as pyrazinacenes1 and porphyrins2 can be incorporated into these structures by different means in particular as MOFs or COFs, while other components include simple transition metal salts or oligonuclear metal-oxo clusters. In this work, we discuss nanohybrids materials containing oxoporphyrinogen (OxP), tetrapyrroles or fullerene as the organic component, with hybridization using respectively Ag(I) salt or oxo-Zr(IV) cluster. This yields nanohybrid materials having structures and properties due to their individual components. Highly-colored OxP is a tetrapyrrole macrocycle which can be stimulated to generate reactive singlet oxygen (1O2) under appropriate conditions (1O2 can be used for applications such as photodynamic therapy and environmental remediation). 1O2 generation by OxP undergoes significant enhancement over its unhybridized state when incorporated in nanohybrid structures, either MOFs or COFs, containing respectively oxo-Zr(IV) nodes or electron deficient linkers in porous coordination polymer nanoarchitectures. OxP-oxoZr(IV) nanohybrid can then be applied for oxidation of small molecule substrates to selected products.3 Also, C60 fullerene can be co-crystallized with silver(I) nitrate at the molecular level4 leading to a material that exhibits unique properties during its decomposition so that silver nitrate is released with concurrent deposition of highly nanostructured C60-only crystalline networks. The resulting nanohybrid material has been studied for application as slow-release antibacterial material.4 Other work includes porous materials for sensing applications. The utility of the nanohybrid approaches is demonstrated by using these examples.

Encapsulation of Pseudomonas aeruginosa elastase inside the P22 virus-like particle for controlling enzyme-substrate interactions.

Controlling interactions between enzymes and interaction partners, such as substrates, is important for applications in cellular biology and molecular biochemistry. A strategy for controlling enzyme access with substrate interaction partners is to exploit encapsulation of enzymes inside nanoparticles to limit the accessibility of the enzymes to large macromolecules, but allow free exchange of small-molecule substrates. The research here evaluates the encapsulation of Pseudomonas aeruginosa elastase inside the bacteriophage P22 virus-like particle (VLP) to examine the ability to allow free soluble substrates access to the enzyme while blocking large macromolecular substrate interactions. The results show that the active elastase protease can be encapsulated inside the P22 VLP, which blocks its ability to disrupt cell monolayers, but allows soluble substrates to be catalytically cleaved, supporting the viability of this approach for future investigations.

Elucidating the mechanisms of the molecular sieving phenomenon created by comb-shaped polymers grafted to a protein – a simulation study

Advances in polymer chemistry now allow the creation of protein–polymer conjugates of great complexity. These advances equally enable the fine-tuning of their structure (and hence properties) to address specific challenges they face as therapeutics. Some of these challenges faced by non-human proteins include their rapid degradation, elimination or immunogenicity in vivo, and one of the most recognized solutions for this is to mask their surface with chains of linear poly(ethylene glycol). This, however, generally reduces bioactivity. Several experimental studies have shown that switching from linear to architecturally complex polymers (comb-shaped, branched, dendronized) partially resolves this issue for a subset of proteins whose bioactivity involves small molecules, by creating a “molecular sieving” effect. The mechanisms underlying molecular sieving, however, have never been entirely elucidated. This study presents a coarse-grained model of α-chymotrypsin modified with multiple chains of the comb-shaped polymer poly(oligo(ethylene glycol) methyl ether methacrylate) to study molecular sieving. Results demonstrate the steric nature of the phenomenon (i.e., creation of gaps in the polymer coating), though steric considerations alone could not reconcile all of the experimental trends. The simulations rather suggest that these gaps enable the selective accumulation of small (substrate) molecules near the surface of the protein (in a favourable microenvironment created by the polymer), which exacerbates the “molecular sieving” phenomenon (i.e., difference in the ability of small vs. large substrates to reach the protein).

Organic Base Enabled Nickel-Catalyzed Mono-α-Arylation of Feedstock Solvents.

We report on our successful development of the first metal-catalyzed mono-α-arylation of carbonyl compounds employing a soluble organic base. The scope of these Ni/DalPhos-catalyzed transformations encompasses a range of (hetero)aryl halides (Cl, Br, I) and phenol-derived electrophiles (sulfonates, carbonates, carbamates, sulfamates), including active pharmaceutical ingredients (chloroquine, clozapine), in combination with the typically problematic feedstock small molecule substrates acetone, dimethylacetamide, and for the first time with any metal catalyst/base, ethyl acetate.

Multiplexed bioluminescence microscopy via phasor analysis

Imaging tools allow us to visualize biological processes, including single cell dynamics and metabolic fluxes. Tracking multiple cells or features over extended periods of time, though, remains challenging. Common approaches rely on fluorescent probes, which require intense excitation light that can damage cells and cause phototoxicity. Bioluminescence imaging (BLI) relies on light production from a chemical reaction, the luciferase-catalyzed oxidation of small molecule substrates (luciferins), and can offer a more bio-friendly alternative for monitoring cellular events in real time.