Phenylalanine Analogues Research Articles

Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002

Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens. Genetic engineering of cyanobacteria conventionally employs antibiotic resistance markers. However, limited availability of antibiotic-resistant markers is a problem for highly multigenic strain engineering. Although several markerless genetic manipulation methods have been developed for PCC 7002, they often lack versatility due to the requirement of gene disruption in the host strain. To achieve markerless transformation in Synechococcus sp. with no requirements for the host strain, this study developed a method in which temporarily introduces a mutated phenylalanyl-tRNA synthetase gene (pheS) into the genome for counter selection. Amino acid substitutions in the PheS that cause high susceptibility of PCC 7002 to the phenylalanine analog p-chlorophenylalanine were examined, and the combination of T261A and A303G was determined as the most suitable mutation. The mutated PheS-based selection was utilized for the markerless knockout of the nblA gene in PCC 7002. In addition, the genetic construct containing the lldD and lldP genes from Escherichia coli was introduced into the ldhA gene site using the counter selection strategy, resulting in a markerless recombinant strain. The repeatability of this method was demonstrated by the double markerless knockin recombinant strain, suggesting it will be a powerful tool for multigenic strain engineering of cyanobacteria.

Direct asymmetric synthesis of β-branched aromatic α-amino acids using engineered phenylalanine ammonia lyases

β-Branched aromatic α-amino acids are valuable building blocks in natural products and pharmaceutically active compounds. However, their chemical or enzymatic synthesis is challenging due to the presence of two stereocenters. We design phenylalanine ammonia lyases (PAL) variants for the direct asymmetric synthesis of β-branched aromatic α-amino acids. Based on extensive computational analyses, we unravel the enigma behind PAL’s inability to accept β-methyl cinnamic acid (β-MeCA) as substrate and achieve the synthesis of the corresponding amino acids of β-MeCA and analogs using a double (PcPAL-L256V-I460V) and a triple mutant (PcPAL-F137V-L256V-I460V). The reactions are scaled-up using an optimized E. coli based whole-cell biotransformation system to produce ten β-branched phenylalanine analogs with high diastereoselectivity (dr > 20:1) and enantioselectivity (ee > 99.5%) in yields ranging from 41-71%. Moreover, we decipher the mechanism of PcPAL-L256V-I460V for the acceptance of β-MeCA and converting it with excellent stereoselectivity by computational simulations. Thus, this study offers an efficient method for synthesizing β-branched aromatic α-amino acids.

Oxidation-Controlled, Strain-Promoted Tellurophene-Alkyne Cycloaddition (OSTAC): A Bioorthogonal Tellurophene-Dependent Conjugation Reaction.

Tellurophene-bearing small molecules have emerged as valuable tools for localizing cellular activities in vivo using mass cytometry. To broaden the utility of tellurophenes in chemical biology, we have developed a bioorthogonal reaction to facilitate tagging of tellurophene-bearing conjugates for downstream applications. Using TePhe, a tellurophene-based phenylalanine analogue, labeled recombinant proteins were generated for reaction development. Using these proteins, we demonstrate an oxidation-controlled, strain-promoted tellurophene-alkyne cycloaddition (OSTAC) reaction. Mild oxidation of the tellurophene ring with N-chlorosuccinimide produces a reactive Te(IV) species which undergoes rapid (k > 100 M-1 s-1) cycloaddition with bicyclo[6.1.0]nonyne (BCN) yielding a benzo-fused cyclooctane. Selective labeling of TePhe-containing proteins can be achieved in complex protein mixtures and on fixed cells. OSTAC reactions can be combined with strain-promoted azide alkyne cycloaddition (SPAAC) and copper-catalyzed azide alkyne click (CuAAC) reactions. Demonstrating the versatility of this approach, we observe the expected staining patterns for 5-ethynyl-2'-deoxyuridine (DNA synthesis-CuAAC) and immunohistochemistry targets in combination with TePhe (protein synthesis-OSTAC) in fixed cells. The favorable properties of the OSTAC reaction suggest its broad applicability in chemical biology.

A convenient broad-host counterselectable system endowing rapid genetic manipulations of Kluyveromyces lactis and other yeast species

Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes.

Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation

BackgroundThe use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties.ResultsIn this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv.ConclusionsThe antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.

Synthesis of β‐Aryl‐α,β‐Dehydroaminophosphonates by Pd‐Catalyzed Fujiwara–Moritani C−C Coupling

AbstractThe treatment of diethyl α,β‐dehydroaminophosphonate 1 with various arenes (ArH=toluene 2 a, benzene 2 b, anisole 2 c, bromobenzene 2 d, chlorobenzene 2 e, benzyl alcohol 2 f, p‐xylene 2 g) in acetic acid (AcOH) at 120 °C for 8 h in the presence of Pd(OAc)2 (10 % mol, OAc=acetate) and AgOAc (3.4 equivalents) results in the formation of the corresponding β‐aryl derivatives E‐Ar(H)C=C(NHAc)P(O)(OEt)2 3 a–3 g. The reaction proceeds through double C−H activation (arene and alkene) and subsequent C−C oxidative coupling (Fujiwara–Moritani reaction), processes catalyzed by Pd and assisted by Ag. The obtained products 3 a–3 g are isosteric analogs of phenylalanine and are obtained with high selectivity. Thus, geometrical E‐isomers have been obtained in all studied cases, however mixtures of ortho‐/meta‐/para‐isomers are observed when the activated position in the starting arene 2 is considered.

Structure–activity characteristics of phenylalanine analogs selectively transported by L-type amino acid transporter 1 (LAT1)

L-type amino acid transporter 1 (LAT1) is a transmembrane protein responsible for transporting large neutral amino acids. While numerous LAT1-targeted compound delivery for the brain and tumors have been investigated, their LAT1 selectivity often remains ambiguous despite high LAT1 affinity. This study assessed the LAT1 selectivity of phenylalanine (Phe) analogs, focusing on their structure–activity characteristics. We discovered that 2-iodo-l-phenylalanine (2-I-Phe), with an iodine substituent at position 2 in the benzene ring, markedly improves LAT1 affinity and selectivity compared to parent amino acid Phe, albeit at the cost of reduced transport velocity. l-Phenylglycine (Phg), one carbon shorter than Phe, was found to be a substrate for LAT1 with a lower affinity, exhibiting a low level of selectivity for LAT1 equivalent to Phe. Notably, (R)-2-amino-1,2,3,4-tetrahydro-2-naphthoic acid (bicyclic-Phe), with an α-methylene moiety akin to the α-methyl group in α-methyl-l-phenylalanine (α-methyl-Phe), a known LAT1-selective compound, showed similar LAT1 transport maximal velocity to α-methyl-Phe, but with higher LAT1 affinity and selectivity. In vivo studies revealed tumor-specific accumulation of bicyclic-Phe, underscoring the importance of LAT1-selectivity in targeted delivery. These findings emphasize the potential of bicyclic-Phe as a promising LAT1-selective component, providing a basis for the development of LAT1-targeting compounds based on its structural framework.

Enhanced anti-tumor activity of arginine decarboxylase through the incorporation of aromatic amino acids at the multimer-forming interface.

The pressing challenge of cancer's high mortality and invasiveness demands improved therapeutic approaches. Targeting the nutrient dependencies within cancer cells has emerged as a promising approach. This study is dedicated to demonstrating the potential of arginine depletion for cancer treatment. Notably, the focus centers on arginine decarboxylase (RDC), a pH-dependent enzyme expecting enhanced activity within the slightly acidic microenvironments of tumors. To investigate the effect of a single-site mutation on the catalytic efficacy of RDC, diverse amino acids, including glycine, alanine, phenylalanine, tyrosine, tryptophan, p-azido-phenylalanine, and a phenylalanine analog with a hydrogen-substituted tetrazine, were introduced at the crucial threonine site (position 39) in the multimer-forming interface. Remarkably, the introduction of either a natural or a non-natural aromatic amino acid at position 39 substantially boosted enzymatic activity, while amino acids with smaller side chains did not show the same effect. This enhanced enzymatic activity is likely attributed to the reinforced formation of multimer structures through favorable interactions between the introduced aromatic amino acid and the neighboring subunit. Noteworthy, at slightly acidic pH, the RDC variant featuring tryptophan at position 39 demonstrated augmented cytotoxicity against tumor cells compared to the wild-type RDC. This attribute aligns with the tumor microenvironment and positions these variants as potential candidates for targeted cancer therapy.

Ancestral l-amino acid oxidase: from substrate scope exploration to phenylalanine ammonia-lyase assay.

In this study we assessed the applicability of the recently reported ancestral l-amino acid oxidase (AncLAAO), for the development of an enzyme-coupled phenylalanine ammonia-lyase (PAL) activity assay. Firstly, the expression and isolation of the AncLAAO-N1 was optimized, followed by activity tests of the obtained octameric N-terminal His-tagged enzyme towards various phenylalanine analogues to assess the compatibility of its substrate scope with that of the well-characterized PALs. AncLAAO-N1 showed high catalytic efficiency towards phenylalanines mono-, di-, or multiple-substituted in the meta- or para-positions, with ortho- substituted substrates being poorly transformed, these results highlighting the significant overlap between its substrate scope and those of PALs. After successful set-up of the AncLAAO-PAL coupled solid phase assay, in a 'proof of concept' approach we demonstrated its applicability for the high-throughput activity screens of PAL-libraries, by screening the saturation mutagenesis-derived I460NNK variant library of PAL from Petroselinum crispum, using p-MeO-phenylalanine as model substrate. Notably, the hits revealed by the coupled assay comprised all the active PAL variants: I460V, I460T, I460S, I460L, previously identified from the tested PAL-library by other assays. Our results validate the applicability of AncLAAO for coupled enzyme systems with phenylalanine ammonia-lyases, including cell-based assays suitable for the high-throughput screening of directed evolution-derived PAL-libraries.

Molecular Syringe for Cargo Photorelease: Red-Light-Triggered Supramolecular Hydrogel.

Photochromic supramolecular hydrogels are versatile materials that show macroscopic effects upon irradiation, like liquefaction or shape changes. Here, we demonstrate a simple photochromic cyclic dipeptide (2,5-diketopiperazine-based) supergelator, composed of (S)-lysine and an azobenzene analogue of phenylalanine, that forms supramolecular hydrogels even at 0.1 wt% loading. The gels can physically encapsulate cargo molecules and release them to the environment in a controllable manner upon irradiation with red light, thus working as a "molecular syringe". As the material is biocompatible and operational in the "therapeutic window" of light (>650 nm) that deeply penetrates soft human tissues, it is applicable to smart drug-delivery systems.

Development of a counterselectable system for rapid and efficient CRISPR-based genome engineering in Zymomonas mobilis

BackgroundZymomonas mobilis is an important industrial bacterium ideal for biorefinery and synthetic biology studies. High-throughput CRISPR-based genome editing technologies have been developed to enable targeted engineering of genes and hence metabolic pathways in the model ZM4 strain, expediting the exploitation of this biofuel-producing strain as a cell factory for sustainable chemicals, proteins and biofuels production. As these technologies mainly take plasmid-based strategies, their applications would be impeded due to the fact that curing of the extremely stable plasmids is laborious and inefficient. Whilst counterselection markers have been proven to be efficient for plasmid curing, hitherto only very few counterselection markers have been available for Z. mobilis.ResultsWe constructed a conditional lethal mutant of the pheS gene of Z. mobilis ZM4, clmPheS, containing T263A and A318G substitutions and coding for a mutated alpha-subunit of phenylalanyl-tRNA synthetase to allow for the incorporation of a toxic analog of phenylalanine, p-chloro-phenylalanine (4-CP), into proteins, and hence leading to inhibition of cell growth. We demonstrated that expression of clmPheS driven by a strong Pgap promoter from a plasmid could render the Z. mobilis ZM4 cells sufficient sensitivity to 4-CP. The clmPheS-expressing cells were assayed to be extremely sensitive to 0.2 mM 4-CP. Subsequently, the clmPheS-assisted counterselection endowed fast curing of genome engineering plasmids immediately after obtaining the desired mutants, shortening the time of every two rounds of multiplex chromosome editing by at least 9 days, and enabled the development of a strategy for scarless modification of the native Z. mobilis ZM4 plasmids.ConclusionsThis study developed a strategy, coupling an endogenous CRISPR-based genome editing toolkit with a counterselection marker created here, for rapid and efficient multi-round multiplex editing of the chromosome, as well as scarless modification of the native plasmids, providing an improved genome engineering toolkit for Z. mobilis and an important reference to develope similar genetic manipulation systems in other non-model organisms.

Machine Learning Enables Prediction of Pyrrolysyl-tRNA Synthetase Substrate Specificity.

Knowledge about the substrate scope for a given enzyme is informative for elucidating biochemical pathways and also for expanding applications of the enzyme. However, no general methods are available to accurately predict the substrate specificity of an enzyme. Pyrrolysyl-tRNA synthetase (PylRS) is a powerful tool for incorporating various noncanonical amino acids (NCAAs) into proteins, which enabled us to probe, image, rationally engineer, and evolve protein structure and function. However, the incorporation of a new NCAA typically requires the selection of large libraries of PylRS with randomized mutations at active sites, and this process requires multiple rounds of selection for each new substrate. Therefore, a single aminoacyl-tRNA synthetase with broad substrate promiscuity is ideal to facilitate widespread applications of the genetic NCAA incorporation technique. Herein, machine learning models were developed to predict the substrate specificity of PylRS to accept novel NCAAs that could be incorporated into proteins by three PylRS mutants. The models were built from a training set of 285 unique enzyme-substrate pairs of three PylRS mutants including IFRS, BtaRS, and MFRS against 95 NCAAs. The best BaggingTree (BT) model was then used for virtually screening a NCAAs library containing 1474 phenylalanine, tyrosine, tryptophan, and alanine analogues, and 156 NCAAs were predicted to be accepted by at least one of the three PylRS mutants. Then, 27 NCAAs including 24 positive and 3 negative substrates were experimentally tested for their activities, and 20 of the 24 positive substrates showed weak or strong activity and were accepted by at least one PylRS mutant, among which 11 NCAAs were never reported to be incorporated into proteins before. Three negative substrates did not show any activity. Experimental results suggested that the BT model provides a three-class classification accuracy of 0.69 and a binary classification accuracy of 0.86. This study expanded the substrate scope of three PylRS variants and provided a framework for developing machine learning models to predict substrate specificity of other PylRS variants.

Sustainable synthesis of L-phenylalanine derivatives in continuous flow by immobilized phenylalanine ammonia lyase

The application of phenylalanine ammonia lyases (PALs) for the amination of a variety of cinnamic acids has been shown to be a cost-efficient method to produce a variety of phenylalanine analogues. Nonetheless, as many other biocatalytic tools, the process intensification, especially due to the high equivalents of ammonia needed, and the cost-efficiency of the catalyst production and use have been key points to further prove their usefulness. Here, we investigated the use of previously characterized PALs (AvPAL and PbPAL) for the amination of a series of substituted cinnamic acids. To enhance the process scalability and the reusability of the catalyst, we investigated the use of covalent immobilization onto commercially available supports, creating a heterogeneous catalyst with good recovered activity (50%) and excellent stability. The immobilized enzyme was also incorporated in continuous flow for the synthesis of 3-methoxy-phenyl alanine and 4-nitro-phenylalanine, which allowed for shorter reaction times (20 min of contact time) and excellent conversions (88% ± 4% and 89% ± 5%) respectively, which could be maintained over extended period of time, up to 24 h. This work exemplifies the advantages that the combination of enzyme catalysis with flow technologies can have not only in the reaction kinetics, but also in the productivity, catalyst reusability and downstream processing.

Highly Effective Markerless Genetic Manipulation of Streptococcus suis Using a Mutated PheS-Based Counterselectable Marker.

Streptococcus suis is an important zoonotic pathogen, however, an efficient markerless genetic manipulation system is still lacking for further physiological and pathological studies on this bacterium. Several techniques have been developed for markerless genetic manipulation of S. suis utilizing either a temperature-sensitive vector or a counterselectable markers (CSMs), however, at present, the efficiency of these techniques is not very satisfactory. In this study, we developed a strategy for markerless genetic manipulation of S. suis employing a CSM based on a conditionally lethal mutant allele of pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase (PheS). This mutant pheS, mPheS, was constructed by introducing site-directed mutations for a T261S/A315G double-substitution and a number of silent mutations to decrease its similarity with the endogenous wild type pheS gene (wtPheS). Additionally, five potentially strong promoters from S. suis were screened for their ability to drive high-level expression of mPheS, thus endowing the carrier strain with sufficient sensitivity to the phenylalanine analog p-chloro-phenylalanine (p-Cl-phe). Insertion of these P-mPheS cassettes into a vector or into the chromosomal locus via a linked erythromycin resistance gene revealed that mPheS allele driven by promoters P0530 and P1503 renders S. suis sensitive to as low as 0.01% (or 0.5 mM) of p-Cl-phe. This offers two potential CSMs for S. suis with p-Cl-phe as a counterselective agent. P1503-mPheS was revealed to be 100% efficient for counter-selection in S. suis by application in a precise gene deletion. Using P1503-mPheS as a CSM, a two-step insertion and excision strategy for markerless genetic manipulation of S. suis were developed, supplying a powerful tool for markerless genetic manipulation of S. suis.

One-pot biosynthesis of aromatic D-amino acids and neuroactive monoamines via enantioselective decarboxylation under in situ product removal using ion exchange resin

Living organisms have evolved a myriad number of enzymes, which inspires reaction engineers to reroute native biocatalytic utility into scalable synthetic purposes. One of such examples is aromatic L-amino acid decarboxylase (AADC), displaying several enzymatic properties desirable for synthetic applications, including a high turnover rate for C-C bond cleavage and strict enantioselectivity. However, exploitation of the catalytic potential has been limited to the preparation of monoamines and has not yet been proven to afford the synthesis of chiral compounds. Here we report AADC-catalyzed kinetic resolution of diverse aromatic amino acids to demonstrate one-pot production of enantiopure D-amino acids as well as neuroactive monoamines. To this end, we used AADC from Bacillus atrophaeus (AADC-BA) displaying broad substrate specificity and excellent enantioselectivity. Kinetic resolution using AADC-BA afforded > 99% ee of D-amino acid leftover at ≈ 50% conversion of racemic substrates such as phenylalanine, tryptophan, tyrosine, homophenylalanine, and substituted phenylalanine analogues. Product inhibition of AADC-BA led us to develop in situ product removal (ISPR) using cation exchange resin, demonstrating that the ISPR strategy facilitated the kinetic resolution owing to selective removal of the inhibitory monoamine. We also demonstrated that low solubility of racemic substrate could be overcome by fed-batch operation.

Regional Brain Analysis of Modified Amino Acids and Dipeptides during the Sleep/Wake Cycle.

Sleep is a state in which important restorative and anabolic processes occur. Understanding changes of these metabolic processes during the circadian rhythm in the brain is crucial to elucidate neurophysiological mechanisms important for sleep function. Investigation of amino acid modifications and dipeptides has recently emerged as a valuable approach in the metabolic profiling of the central nervous system. Nonetheless, very little is known about the effects of sleep on the brain levels of amino acid analogues. In the present study, we examined brain regional sleep-induced alterations selective for modified amino acids and dipeptides using Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) based metabolomics. Our approach enabled the detection and identification of numerous amino acid-containing metabolites in the cortex, the hippocampus, the midbrain, and the cerebellum. In particular, analogues of the aromatic amino acids phenylalanine, tyrosine and tryptophan were significantly altered during sleep in the investigated brain regions. Cortical levels of medium and long chain N-acyl glycines were higher during sleep. Regional specific changes were also detected, especially related to tyrosine analogues in the hippocampus and the cerebellum. Our findings demonstrate a strong correlation between circadian rhythms and amino acid metabolism specific for different brain regions that provide previously unknown insights in brain metabolism.

Reagentless electrochemical biosensors through incorporation of unnatural amino acids on the protein structure

Typical protein biosensors employ chemical or genetic labeling of the protein, thus introducing an extraneous molecule to the wild-type parent protein, often changing the overall structure and properties of the protein. While these labeling methods have proven successful in many cases, they also have a series of disadvantages associated with their preparation and function. An alternative route for labeling proteins is the incorporation of unnatural amino acid (UAA) analogues, capable of acting as a label, into the structure of a protein. Such an approach, while changing the local microenvironment, poses less of a burden on the overall structure of the protein. L-DOPA is an analog of phenylalanine and contains a catechol moiety that participates in a quasi-reversible, two-electron redox process, thus making it suitable as an electrochemical label/reporter. The periplasmic glucose/galactose binding protein (GBP) was chosen to demonstrate this detection principle. Upon glucose binding, GBP undergoes a significant conformational change that is manifested as a change in the electrochemistry of L-DOPA. The electroactive GBP was immobilized onto gold nanoparticle-modified, polymerized caffeic acid, screen-printed carbon electrodes (GBP-LDOPA/AuNP/PCA/SPCE) for the purpose of direct measurement of glucose levels and serves as a proof-of-concept of the use of electrochemically-active unnatural amino acids as the label. The resulting reagentless GBP biosensors exhibited a highly selective and sensitive binding affinity for glucose in the micromolar range, laying the foundation for a new biosensing methodology based on global incorporation of an electroactive amino acid into the protein's primary sequence for highly selective electrochemical detection of compounds of interest.

Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase.

This study reports the application of expanding genetic codes in developing protein cage-based delivery systems. The evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pairs derived from directed evolution are examined to probe their recognition for para-substituted phenylalanine analogs. The evolved MmPylRS, AzFRS, harboring a wide range of substrates, is further engineered at the C-terminal region into another variant, AzFRS-MS. AzFRS-MS shows suppression of the elevated sfGFP protein amount up to 10 TAG stop codons when charging p-azido-l-phenylalanine (AzF, 4), which allows the occurrence of click chemistry. Since protein nanocages used as drug delivery systems that encompass multiple drugs through a site-specific loading approach remain largely unexplored, as a proof of concept, the application of AzFRS-MS for the site-specific incorporation of AzF on human heavy chain ferritin (Ftn) is developed. The Ftn-4 conjugate is shown to be able to load multiple fluorescence dyes or a therapeutic agent, doxorubicin (Dox), through the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Aiming to selectively target Her2+ breast cancer cells, Ftn-4-DOX conjugates fused with a HER2 receptor recognition peptide, anti-Her2/neu peptide (AHNP), is developed and demonstrated to be able to deliver Dox into the cell and to prolong the drug release. This work presents another application of evolved MmPylRS systems, whose potential in developing a variety of protein conjugates is noteworthy.

Identification of Antagonistic Compounds between the Palm Tree Xylariale Endophytic Fungi and the Phytopathogen Fusarium oxysporum.

To discover microorganisms that naturally possess chemical weapons against the phytopathogen Fusarium oxysporum, the biological and chemical diversity of plant leaf endophytes was investigated. Endophytes were isolated from the palm tree Astrocaryum sciophyllum collected in pristine forests of French Guiana. Several Xylariaceae inhibited the growth of F. oxysporum and were further explored. Antifungal specialized metabolites were isolated from the Xylariaceae BSNB-0294 strain in confrontation with the phytopathogen and led to the identification of undescribed compounds, i.e., two depsipeptides named xylariaceins, two metabolites containing a 3-imidazolinone moiety, and four new compounds including a nitro-phenylpropanamide and three phenylalanine analogues named xylariains A-D. In parallel, the chemical investigation of the phytopathogen during the coculture led to the identification of an unknown compound, which we named focicin. The production of focicin was exacerbated during the competition. Matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectometry (MALDI-TOF MS) imaging of the competition between BSNB-0294 (endophytic strain) and F. oxysporum f.sp. ciceris (phytopathogen) highlighted time-dependent chemical interactions between the two microorganisms.

Decreased proteasomal cleavage at nitrotyrosine sites in proteins and peptides

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.