N-terminal Position Research Articles

Anti-Inflammatory Effect of Homo- and Heterodimers of Natural Enkephalinase Inhibitors in Experimental Colitis in Mice.

Background: the pharmacological treatment and/or maintenance of remission in inflammatory bowel diseases (IBDs) is currently one of the biggest challenges in the field of gastroenterology. Method: our aim was the synthesis of homo- and heterodimers of natural enkephalinase inhibitors (opiorphin; sialorphin; spinorphin) and the in vitro characterization of their effect on the degradation of enkephalin by neutral endopeptidase (NEP) and stability in human plasma. We investigated the in vivo heterodimer of Cys containing analogs of sialorphin and spinorphin (peptide X) in a mouse model of colitis. The extent of inflammation was evaluated based on the microscopic score; macroscopic score; ulcer score, colonic wall thickness, colon length and quantification of myeloperoxidase activity. Results: we showed that the homo- and heterodimerization of analogs of sialorphin, spinorphin and opiorphin containing Cys residue at the N-terminal position resulted in dimeric forms which in vitro exhibited higher inhibitory activity against NEP than their parent and monomeric forms. We showed that peptide X was more stable in human plasma than sialorphin and spinorphin. Peptide X exerts potent anti-inflammatory effect in the mouse model of colitis. Conclusion: we suggest that peptide X has the potential to become a valuable template for anti-inflammatory therapeutics for the treatment of gastrointestinal (GI) tract inflammation.

Heterologous Biosynthesis of Five New Class II Bacteriocins From Lactobacillus paracasei CNCM I-5369 With Antagonistic Activity Against Pathogenic Escherichia coli Strains.

Lactobacillus paracasei CNCM I-5369 isolated from a traditional Algerian dairy product produces extracellular inhibitory substances, namely, bacteriocins, which are active against a panel of pathogenic Escherichia coli strains. This activity was observed only at a narrow pH 4.5–5, and resulted to be heat stable and sensitive to the action of proteolytic enzymes, which indicate a proteinaceous nature. This new strain has a genome of 2,752,975 bp, with a 46.6% G + C ratio and contains at least 2664 coding sequences. The Bagel software analysis identified five open reading frames (ORFs) that are translated to new class II bacteriocin. Each ORF was cloned in frame with a His-tag tail and expressed in E. coli BL21 (DE3) (pLysS) strain. Of note, each fusion protein carrying any of these ORFs at the C- or N-terminal position resulted to be active against E. coli 184 strain used as target organism. This manuscript reports the first multi-bacteriocinogenic strain producing five new class II bacteriocins with activity against Gram-negative bacilli (GNB), namely, E. coli. Heterologous expression and activity of each new class II bacteriocin were demonstrated.

Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching.

A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.

Structure-activity relationships of tubulysin analogues

The tubulysins are an emerging antibody-drug conjugate (ADC) payload that maintain potent anti-proliferative activity against cells that exhibit the multi-drug resistant (MDR) phenotype. These drugs possess a C-11 acetate known to be hydrolytically unstable in plasma, and loss of the acetate significantly attenuates cytotoxicity. Structure-activity relationship studies were undertaken to identify stable C-11 tubulysin analogues that maintain affinity for tubulin and potent cytotoxicity. After identifying several C-11 alkoxy analogues that possess comparable biological activity to tubulysin M with significantly improved plasma stability, additional analogues of both the Ile residue and N-terminal position were synthesized. These studies revealed that minor changes within the tubulin binding site of tubulysin can profoundly alter the activity of this chemotype, particularly against MDR-positive cell types.

An In Vivo Targeted Deletion of the Calmodulin-Binding Domain from Rice Glutamate Decarboxylase 3 (OsGAD3) Increases \u03b3-Aminobutyric Acid Content in Grains

BackgroundGamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). In plants the enzymatic activity of GAD is activated by Ca2+/calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice.ResultsIn this study, we focused on GAD3, one of five GAD genes present in the rice genome, because GAD3 is the predominantly expressed in seeds, as reported previously. We confirmed that GAD3 has an authentic Ca2+/CaMBD that functions as an autoinhibitory domain. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the OsGAD3 gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via Agrobacterium to regenerate rice plants. Out of 24 transformed rice (T1), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding OsGAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a AKNQDAAD peptide in the C-terminal region of the putative OsGAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). A very similar rice line (#8_1) carrying AKNRSSRRSGR in OsGAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Free amino acid analysis of the seeds (T2) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca2+/bovine CaM were both higher than wild-type counterpart. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1.ConclusionsWe have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. This is the first report of the production of GABA-enriched rice via a genome editing.

Ultrashort Cationic Lipopeptides-Effect of N-Terminal Amino Acid and Fatty Acid Type on Antimicrobial Activity and Hemolysis.

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that hypothetically may be alternatively used to combat pathogens such as bacteria and fungi. In general, USCLs consist of fatty acid chains and a few basic amino acid residues. The main shortcoming of USCLs is their relatively high cytotoxicity and hemolytic activity. This study focuses on the impact of the hydrophobic fatty acid chain, on both antimicrobial and hemolytic activities. To learn more about this region, a series of USCLs with different straight-chain fatty acids (C8, C10, C12, C14) attached to the tripeptide with two arginine residues were synthesized. The amino acid at the N-terminal position was exchanged for proteinogenic and non-proteinogenic amino acid residues (24 in total). Moreover, the branched fatty acid residues were conjugated to N-terminus of a dipeptide with two arginine residues. All USCLs had C-terminal amides. USCLs were tested against reference bacterial strains (including ESKAPE group) and Candida albicans. The hemolytic potential was tested on human erythrocytes. Hydrophobicity of the compounds was evaluated by RP-HPLC. Shortening of the fatty acid chain and simultaneous addition of amino acid residue at N-terminus were expected to result in more selective and active compounds than those of the reference lipopeptides with similar lipophilicity. Hypothetically, this approach would also be beneficial to other antimicrobial peptides where N-lipidation strategy was used to improve their biological characteristics.

N-terminal sequences affect expression of triterpene biosynthesis enzymes in Chlamydomonas chloroplasts

Metabolic engineering is an emerging technology to modify the biochemical properties of living cells. In microalgae, metabolic engineering has often been directed towards optimizing the production of desirable lipids or related bioproducts. Here we describe efforts to engineer the green alga Chlamydomonas reinhardtii for the production of botryococcene, a drop-in biofuel precursor. Genes encoding farnesyl diphosphate synthase (FPS) and squalene synthase-like (SSL)-1 and -3, were introduced into the chloroplast genome using biolistic transformation. Through a series of construct modifications, we identified intergenic sequences that promote expression of stable, discrete transcripts. We also found amino acids that dramatically increased the accumulation of SSL-3 when they were inserted at the N-terminal penultimate position, and similar manipulation of the N-terminal sequence of FPS appeared to improve its protein level as well. However, SSL-1 only accumulated to detectable levels when expressed as a chimera with SSL-3. In vitro assays showed that chloroplast-expressed SSL-3 was enzymatically active, but not SSL-1, although the SSL-1-SSL-3 chimeras were active when expressed in yeast. Taken together, our results suggest that the N-terminal sequence and other cellular factors are important when heterologous proteins are expressed in this model algal species.

Fibrous Aggregates of Short Peptides Containing Two Distinct Aromatic Amino Acid Residues.

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C-terminal, N-terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H-PheCysTrp-OH, H-PheCysTyr-OH, H-PheTyrMet-OH, and H-TrpTyr-OH.

Site-Selective Protein Immobilization on Polymeric Supports through N-Terminal Imidazolidinone Formation.

Protein immobilization techniques on polymeric supports have enabled many applications in biotechnology and materials science. Attaching the proteins with controlled orientations has inherent advantages, but approaches for doing this have been largely limited to cysteine or noncanonical amino acid targeting. Herein, we report a method to attach the N-terminal positions of native proteins to polymer resins site-specifically through the use of 2-pyridinecarboxyaldehyde (2PCA) derivatives. For high protein loadings and practical synthesis, we initiated this work by preparing highly reactive 2PCA derivatives using Pd-catalyzed cross-coupling amination. The resulting compounds were attached to amine-containing polyethylene glycol acrylamide resin (PEGA-NH2), which subsequently reacted with the N-termini of proteins to produce linkages that were stable over the long term but could be reversed through the addition of hydroxylamine. We envision that this site-selective, 2PCA-based protein immobilization can provide a simple and generalizable immobilization protocol.

Dimeric C34 Derivatives Linked through Disulfide Bridges as New HIV-1 Fusion Inhibitors.

C34, a 34-mer fragment peptide, is contained in the HIV-1 envelope protein gp41. A dimeric derivative of C34 linked through a disulfide bridge at its C terminus was synthesized and found to display potent anti-HIV activity, comparable with that of a previously reported PEGylated dimer of C34REG. The reduction in the size of the linker moiety for dimerization was thus successful, and this result might shed some light on the mechanism of the suppression of six-helix bundle formation by these C34 dimeric derivatives. Addition of a Gly-Cys(CH2 CONH2 )-Gly-Gly motif at the N-terminal position of a C34 monomeric derivative significantly increased the anti-HIV-1 activity. This moiety functions as a new pharmacophore, and this might provide a useful insight into the design of potent HIV-1 fusion inhibitors.

Mechanisms of Neuroprotective Effects of Peptides Derived from Natural Materials and Their Production and Assessment.

In this review, we provide an overview of the main mechanisms by which peptides derived from natural materials exhibit neuroprotective effects. We also review methods for the production of these peptides and various methods for determining their neuroprotective effects in vitro and in vivo. Neuroprotective peptides are closely associated with protection against apoptosis, with proteins related to the cell death/survival signaling pathway, with acetylcholine activity and with inhibition of oxidative stress and inflammation. Neuroprotective peptides derived from natural materials typically have a molecular weight below 1 kDa; they are most often derived using the extraction enzymes papain and alcalase. The neuroprotective peptides identified in the literature are mainly composed of hydrophobic amino acids with low molecular weight, with Asp and Glu at the N-terminal position. These observations may guide the development of novel peptides with potential neuroprotective effects.

N-degron specificity of chloroplast ClpS1 in plants.

The prokaryotic N-degron pathway depends on the Clp chaperone-protease system and the ClpS adaptor for recognition of N-degron bearing substrates. Plant chloroplasts contain a diversified Clp protease, including the ClpS homolog ClpS1. Several candidate ClpS1 substrates have been identified, but the N-degron specificity is unclear. Here, we employed invitro ClpS1 affinity assays using eight N-degron green fluorescence protein reporters containing either F, Y, L, W, I, or R in the N-terminal position. This demonstrated that ClpS1 has a restricted N-degron specificity, recognizing proteins bearing an N-terminal F or W, only weakly recognizing L, but not recognizing Y or I. This affinity is dependent on two conserved residues in the ClpS1 binding pocket and is sensitive to FR dipeptide competition, suggesting a unique chloroplast N-degron pathway.

Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives.

A convenient enzymatic strategy is reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus (abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepared on large scale if needed, (2) mild reaction conditions, including low temperatures, (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of molecular oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo molecules to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction.

Engineering ClpS for selective and enhanced N-terminal amino acid binding.

One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity sequential recognition and detection of specific amino acids that comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-end rule pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the Agrobacterium tumefaciens ClpS protein with native residues 34-36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.

Cytosolic Delivery of Proteins Using Amphiphilic Polymers with 2-Pyridinecarboxaldehyde Groups for Site-Selective Attachment

Protein-based drugs are a promising class of therapeutics, but poor membrane permeability typically limits their application to extracellular receptors. Delivery strategies that can transport functional proteins to reach intracellular targets are needed, but with many current approaches, biomolecules become entrapped in the endosomes. This greatly reduces the effective concentrations of therapeutic agents at the target sites. Herein, we report a bioconjugation-based approach for intracellular protein delivery by site-selectively attaching amphiphilic polymers to the N-terminal positions of proteins using 2-pyridinecarboxaldehyde groups. The reaction is simple and features mild, aqueous conditions with no required genetic engineering of the proteins. Imaging studies demonstrate that the polymer-protein conjugates are successfully delivered into the cytosol of various cancer cell lines, likely through a membrane fusion mechanism. When conjugated to the delivery polymers, the activity of modified RNase A was retained and notably promoted cytotoxicity in cancer cells upon delivery to the cytosol. This work therefore provides a promising platform for protein-based material delivery for therapeutic applications.

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α-position and peptides on N-terminal position.

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N-α or ε-acetyl-L-lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε-position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N-acylation on the α-position of the lysine or of the amino-acid in N-terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N-α-acetyl-L-lysine could be attributed to the product of SAM23877_1734 gene.

Search for Fibrous Aggregates Potentially Useful in Regenerative Medicine Formed under Physiological Conditions by Self-Assembling Short Peptides Containing Two Identical Aromatic Amino Acid Residues.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H-dPhedPhe-OH, H-dCysdPhedPhe-OH, H-CysTyrTyr-OH, H-dPhedPhedCys-OH, H-TyrTyrMet-OH, and H–TyrMetTyr–OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

Analysis of essential gene dynamics under antibiotic stress in Streptococcus sanguinis.

The paradoxical response of Streptococcus sanguinis to drugs prescribed for dental and clinical practices has complicated treatment guidelines and raised the need for further investigation. We conducted a high throughput study on concomitant transcriptome and proteome dynamics in a time course to assess S. sanguinis behaviour under a sub-inhibitory concentration of ampicillin. Temporal changes at the transcriptome and proteome level were monitored to cover essential genes and proteins over a physiological map of intricate pathways. Our findings revealed that translation was the functional category in S. sanguinis that was most enriched in essential proteins. Moreover, essential proteins in this category demonstrated the greatest conservation across 2774 bacterial proteomes, in comparison to other essential functional categories like cell wall biosynthesis and energy production. In comparison to non-essential proteins, essential proteins were less likely to contain ‘degradation-prone’ amino acids at their N-terminal position, suggesting a longer half-life. Despite the ampicillin-induced stress, the transcriptional up-regulation of amino acid-tRNA synthetases and proteomic elevation of amino acid biosynthesis enzymes favoured the enriched components of essential proteins revealing ‘proteomic signatures’ that can be used to bridge the genotype–phenotype gap of S. sanguinis under ampicillin stress. Furthermore, we identified a significant correlation between the levels of mRNA and protein for essential genes and detected essential protein-enriched pathways differentially regulated through a persistent stress response pattern at late time points. We propose that the current findings will help characterize a bacterial model to study the dynamics of essential genes and proteins under clinically relevant stress conditions.

Trifluoromethylated proline analogues as efficient tools to enhance the hydrophobicity and to promote passive diffusion transport of the l-prolyl-l-leucyl glycinamide (PLG) tripeptide.

The synthesis of four CF3-proline analogues of the PLG peptide is reported. Our results show that the incorporation of trifluoromethylated amino acids (Tfm-AAs) at the N-terminal position of a peptide significantly increases its hydrophobicity. In addition, depending on the relative configuration and the position of the CF3 group, Tfm-AAs can also promote passive diffusion transport.

Fusion with the cold-active esterase facilitates autotransporter-based surface display of the 10th human fibronectin domain in Escherichia coli.

Cell surface display is a popular approach for the construction of whole-cell biocatalysts, live vaccines, and screening of combinatorial libraries. To develop a novel surface display system for the popular scaffold protein 10th human fibronectin type III domain (10Fn3) in Escherichia coli cells, we have used an α-helical linker and a C-terminal translocator domain from previously characterized autotransporter from Psychrobacter cryohalolentis K5T. The level of 10Fn3 passenger exposure at the cell surface provided by the hybrid autotransporter Fn877 and its C-terminal variants was low. To improve it, the fusion proteins containing 10Fn3 and the native autotransporter passenger Est877 or the cold-active esterase EstPc in different orientations were constructed and expressed as passenger domains. Using the whole-cell ELISA and activity assays, we have demonstrated that N-terminal position of EstPc in the passenger significantly improves the efficiency of the surface display of 10Fn3 in E. coli cells.