Leader Peptide Sequence Research Articles

Structure of the murine CD94-NKG2A receptor in complex with Qa-1b presenting an MHC-I leader peptide.

The heterodimeric natural killer cells antigen CD94 (CD94)-NKG2-A/NKG2-B type II integral membrane protein (NKG2A) receptor family expressed on human and mouse natural killer (NK) cells monitors global major histocompatibility complex (MHC) class I cell surface expression levels through binding to MHC class Ia-derived leader sequence peptides presented by HLA class I histocompatibility antigen, alpha chain E (HLA-E; in humans) or H-2 class I histocompatibility antigen, D-37 (Qa-1b; in mice). Although the molecular basis underpinning human CD94-NKG2A recognition of HLA-E is known, the equivalent interaction in the murine setting is not. By determining the high-resolution crystal structure of murine CD94-NKG2A in complex with Qa-1b presenting the Qa-1 determinant modifier peptide (QDM), we resolved the mode of binding. Compared to the human homologue, the murine CD94-NKG2A-Qa-1b-QDM displayed alterations in the distribution of interactions across CD94 and NKG2A subunits that coincide with differences in electrostatic complementarity of the ternary complex and the lack of cross-species reactivity. Nevertheless, we show that Qa-1b could be modified through W65R + N73I mutations to mimic HLA-E, facilitating binding with both human and murine CD94-NKG2A. These data underscore human and murine CD94-NKG2A cross-species heterogeneity and provide a foundation for humanising Qa-1b in immune system models.

Exploration, expansion and definition of the atropopeptide family of ribosomally synthesized and posttranslationally modified peptides.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) constitute a diverse class of natural products. Atropopeptides are a recent addition to the class. Here we developed AtropoFinder, a genome mining algorithm to chart the biosynthetic landscape of the atropopeptides. AtropoFinder identified more than 650 atropopeptide biosynthetic gene clusters (BGCs). We pinpointed crucial motifs and residues in leader and core peptide sequences, prompting a refined definition of the atropopeptide RiPP family. Our study revealed that a substantial subset of atropopeptide BGCs harbors multiple tailoring genes, thus suggesting a broader structural diversity than previously anticipated. To verify AtropoFinder, we heterologously expressed four atropopeptide BGCs, which resulted in the identification of novel atropopeptides with varying peptide lengths, number and types of modifications. Atropopeptides serve as a proof-of-principle for the versatile genome mining approach developed in this study that can be repurposed for the identification of RiPP and other BGCs that currently evade detection.

AML Immunotherapy Using a Novel Tcrm-Based Bispecific Antibody That Targets a Leader Sequence Peptide Derived from Cathepsin G

Introduction: Myeloid azurophil granules provide a rich source of intracellular leukemia antigens. Cathepsin G (CG) is a serine protease that has higher expression in AML blasts in comparison to normal myeloid progenitors. Based on the unique biology of HLA-A*0201 (HLA-A2), in which presentation of leader sequence (LS)-derived peptides is favored, we focused on the LS-CG-derived peptide CG1 (FLLPTGAEA). LS peptides are naturally processed and loaded on HLA-A2, providing an abundant source of surface peptide/HLA (pHLA) targets. We eluted CG1 from the surface of primary HLA-A2 + AML blasts and AML cell lines, and demonstrated CG1-targeting immunity in leukemia patients following allogeneic stem cell transplant, hence highlighting the potential for CG1 to be a promising immunotherapeutic target in AML. Numerous platforms have been developed to target cell surface pHLA complexes. T cell receptor (TCR) mimic (m) antibodies are immunotherapeutic antibodies that target pHLA, which are the natural ligands for the TCR. TCRm antibodies have been a breakthrough in immunotherapy, as they: (1) target intracellular antigens expressed on HLA; (2) are engineered to have high affinity for HLA; (3) can elicit their cytotoxic effect independent of the TCR; and (4) can be produced in large quantities providing an off-the shelf-product for patient use. Here we report on the engineering, preclinical efficacy, and safety evaluation of a novel CG1-targeting, T cell-engager, bispecific antibody (CG1xCD3e), which incorporates a novel TCRm construct that recognizes cell surface CG1/HLA-A2 complexes. Methods: To generate the CG1/HLA-A2-binding monoclonal antibodies, H2L2 human transgenic mice were immunized with CG1/HLA-A2 monomers subcutaneously. The resulting paired heavy and light chains were cloned from the antigen-sorted memory B cells into the single pcDNA3.1(+) vector with both human (h) immunoglobulin (Ig)G1 and hKappa for subsequent ExpiCHO high-throughput transient expression. Antibody binding was assessed by Bio-Layer Interferometry (BLI) and T2 cellular binding assays. Xencor anti-CD3e scFv and Knobs-in-holes technology was applied for the bispecific antibody engineering for xMab format. In vitro activity of CG1xCD3e and T cell cytokine secretion was confirmed using flow cytometry-based T cell-dependent cellular cytotoxicity (TDCC). The specificity of CG1xCD3e for leukemia was assessed using colony forming unit assays. In vivo efficacy was evaluated in NSG mice engrafted with AML cell lines and treated with CG1xCD3e with normal donor peripheral blood mononuclear cells (ND-PBMC). Results: Using BLI, binding avidity of the CG1xCD3e to CD3-Fc, KD= 7.52 x 10-10 M, and affinity to CG1/HLA-A2, KD= 1.28 x 10-10 M were calculated. Using flow cytometry, we confirmed high affinity binding of CG1xCD3e to CG1/HLA on the CG1- expressing EM2 HLA-A2 + cell line, and also demonstrated binding of CG1xCD3e to CD3 using jurkat T cell line. After co-culturing GFP/Luciferase + HLA-A2 + AML (U937-A2 and ML-2) or CML (EM2) cell lines with ND-PBMC and different concentrations of CG1xCD3e bispecific antibody for 24 or 48 hours, flow cytometry analysis demonstrated highly specific CG1/HLA-A2 dependent killing of AML cells by T cells cultured with CG1xCD3e bispecific antibody, in comparison with control bispecific antibody with a disabled CG1 binding arm. This correlated with both tumor- and bispecific antibody- dependent T cell activation and cytokine secretion. To study the in vivo anti-leukemia activity of CG1xCD3e, we engrafted NSG mice with the HLA-A2 + ML-2 and U937-A2 AML cell lines, and treated mice after tumor engraftment with ND-PBMC (1 x 107 cells) and weekly intraperitoneal injections of CG1xCD3e bispecific antibody (0.01 mg/Kg, 0.05 mg/Kg, and 0.1 mg/Kg) or PBS. Bioluminescence imaging demonstrated that the mice treated with ND-PBMC + CG1xCD3e had significantly lower levels of leukemia burden compared to the mice treated with ND-PBMC alone (Figure 1). Lastly CFU assays using HLA-A2 + normal donor marrow in a semi-solid matrix of methylcellulose in the presence of T cells and either CG1xCD3e bispecific antibody or isotype antibody, confirmed specificity of CG1xCD3e bispecific antibody for leukemia. Conclusion: Our study provides strong pre-clinical evidence supporting the targeting of LS- derived peptides, specifically CG1, in the setting of AML using a novel TCRm-based bispecific antibody.

Mechanistic Investigations into the Catalytic Mode of a Dehydratase Complex Involved in the Biosynthesis of Lantibiotic Cacaoidin

Comprehensive SummaryDehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme (domain) during the biosynthesis of RiPP. Recently, it was reported that dehydration in the thioviridamide pathway relies on a distinct dehydratase complex that showcases the activities of a phosphotransferase TvaC for serine/threonine phosphorylation and a lyase TvaD for subsequent phosphate elimination. Herein, we report that dehydration reactions in the pathway of lantibiotic cacaoidin involves a similar dehydratase complex, CaoK/CaoY. Remarkably, this dehydratase complex exhibits flexible enzymatic activity and tolerates significant variations in its substrate peptide sequence. By binding with the leader peptide (LP) sequence of precursor peptide CaoA, the dehydration reactions proceed in a directional manner from the C‐terminus of the core peptide (CP) to the N‐terminus, and C‐terminally truncated variants of CP are acceptable. We show that fusing CaoK to CaoY in a 1 : 1 molar ratio enables the resulting enzyme CaoYK to exert enhanced dehydration activity. CaoK binds with the LP to improve its own solubility and to ensure the phosphate transfer activity, while CaoY functions in a manner independently of LP. This work advances our understanding of the dehydration process during cacaoidin formation, and provides useful enzymes and methods for the studies of the rapidly emerging RiPPs.

Insulin and the kidneys: a contemporary view on the molecular basis.

Insulin is a hormone that is composed of 51 amino acids and structurally organized as a hexamer comprising three heterodimers. Insulin is the central hormone involved in the control of glucose and lipid metabolism, aiding in processes such as body homeostasis and cell growth. Insulin is synthesized as a large preprohormone and has a leader sequence or signal peptide that appears to be responsible for transport to the endoplasmic reticulum membranes. The interaction of insulin with the kidneys is a dynamic and multicenter process, as it acts in multiple sites throughout the nephron. Insulin acts on a range of tissues, from the glomerulus to the renal tubule, by modulating different functions such as glomerular filtration, gluconeogenesis, natriuresis, glucose uptake, regulation of ion transport, and the prevention of apoptosis. On the other hand, there is sufficient evidence showing the insulin receptor's involvement in renal functions and its responsibility for the regulation of glucose homeostasis, which enables us to understand its contribution to the insulin resistance phenomenon and its association with the progression of diabetic kidney disease.

Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2.

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

Characterization of a novel therapeutic antibody targeting peptide/HLA-E

Abstract T-cell receptor mimic antibodies (TCRm) have become an attractive therapeutic modality group as they allow for the targeting of intracellular proteins presented by HLA, which may allow for a large potential of new targets. However, the polymorphic nature of classical HLA class I and findings of HLA downregulation in cancer cells might limit the use of these agents for cancer treatment. An alternative approach would be to target peptides presented by HLA-E, a highly monomorphic molecule often found upregulated in cancer cells. Thus, we aimed to generate a TCRm against HLA-E presenting the leader sequence peptide from HLA-G since both HLAs are often expressed in multiple cancer types. An antibody was identified from a naïve human phage library and further affinity matured using yeast display. The final clone, ABX-001, had an affinity constant (K D) of 7.9nM. Specificity studies using both peptide-loaded cells and tumor cell lines showed that ABX-001 only bound when both HLA-E and HLA-G were present. Extensive profiling was done on immune cells to also confirm that they did not bind ABX-001. Finally, ABX-001 was assessed for its ability to direct killing of target-positive tumor cell lines. We observed that cytotoxicity in CD8 +T cells was enhanced with the addition of an ABX-001-CD3 T cell engager. We also observed that ABX-001 can act as a checkpoint inhibitor by blocking the interaction of HLA-E with NKG2A in cytotoxicity experiments using NK cells. These data indicate that we can effectively target peptide/HLA-E and highlights its potential therapeutic applications in cancer treatment.

LSPpred Suite: Tools for Leaderless Secretory Protein Prediction in Plants.

Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives controlled at 5%, and are also able to classify the limited number of established plant LSPs (SPLpred (3/4, LSPpred 4/4). Until such time as a larger set of bona fide (independently experimentally validated) LSPs is established using imaging technologies (light/fluorescence/electron microscopy) to confirm sub-cellular location, these tools represent a bridging method for predicting and identifying plant putative LSPs for subsequent experimental validation.

An antimicrobial thiopeptide producing novel actinomycetes Streptomyces terrae sp. nov., isolated from subsurface soil of arable land.

An antimicrobial producing Gram-positive, aerobic, nonmotile, and filamentous actinobacterial strain SKN60T was isolated from soil The isolate exhibited 99.3% and 99.0% identity with Streptomyces laurentii ATCC 31255T and S. roseicoloratus TRM 44457T, respectively, in 16S rRNA gene sequence analysis. However, the genome sequence displayed maximum ANI (88.45%) and AAI (85.61%) with S. roseicoloratus TRM 44457T. Similarly, the dDDH showed 33.7% identity with S. roseicoloratus TRM 44457T. It formed a cluster with S. roseicoloratus TRM 44457T and S. laurentii ATCC 31255T in phylogenomic tree. Cell wall analysis revealed the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine as major polar lipids and diaminopimelic acid as diagnostic diamino acid. Major fatty acids were iso-C15:0, anteiso-C15:0, and iso-C16:0. The G+C content was found to be 72.3 mol%. Genome sequence analysis using antiSMASH database showed occurrence of a thiopeptide biosynthesis gene cluster with 94% similarity to berninamycin from S. bernensis UC5144. The mass of 1146Da is identical with berninamycin. But subtle differences observed in leader peptide sequence of thiopeptide and berninamycin. Notably, S. bernensis is not validly reported and thus SKN60T is the only strain containing berninamycin BGC as no other phylogenetic relative had it. Additionally, strain SKN60T differed in phenotypic and genetic characteristics with all phylogenetic relatives of the genus Streptomyces. Therefore, it is proposed as a novel species with the name Streptomyces terrae sp. nov. strain SKN60T (=MTCC 13163T; = JCM 35768T).

Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

Human cytomegalovirus (HCMV) infections develop into CMV diseases that result in various forms of manifestations in local organs. CMV-retinitis is a form of CMV disease that develops in immunocompromised hosts with CMV-viremia after viruses in the peripheral circulation have entered the eye. In the HCMV genome, extensive diversification of the UL40 gene has produced peptide sequences that modulate NK cell effector functions when loaded onto HLA-E and are subsequently recognized by the NKG2A and NKG2C receptors. Notably, some HCMV strains carry UL40 genes that encode peptide sequences identical to the signal peptide sequences of specific HLA-A and HLA-C allotypes, which enables these CMV strains to escape HLA-E-restricted CD8+T cell responses. Variations in UL40 sequences have been studied mainly in the peripheral blood of CMV-viremia cases. In this study, we sought to investigate how ocular CMV disease develops from CMV infections. CMV gene sequences were compared between the intraocular fluids and peripheral blood of 77 clinical cases. UL40 signal peptide sequences were more diverse, and multiple sequences were typically present in CMV-viremia blood compared to intraocular fluid. Significantly stronger NK cell suppression was induced by UL40-derived peptides from intraocular HCMV compared to those identified only in peripheral blood. HCMV present in intraocular fluids were limited to those carrying a UL40 peptide sequence corresponding to the leader peptide sequence of the host's HLA class I, while UL40-derived peptides from HCMV found only in the peripheral blood were disparate from any HLA class I allotype. Overall, our analyses of CMV-retinitis inferred that specific HCMV strains with UL40 signal sequences matching the host's HLA signal peptide sequences were those that crossed the blood-ocular barrier to enter the intraocular space. UL40 peptide repertoires were the same in the intraocular fluids of all ocular CMV diseases, regardless of host immune status, implying that virus type is likely to be a common determinant in ocular CMV disease development. We thus propose a mechanism for ocular CMV disease development, in which particular HCMV types in the blood exploit peripheral and central HLA-E-mediated tolerance mechanisms and, thus, escape the antivirus responses of both innate and adaptive immunity.

Characterization of Histidine Functionalization and Its Timing in the Biosynthesis of Ribosomally Synthesized and Posttranslationally Modified Thioamitides.

Thioamitides are ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products that hold great potential in anticancer drug development. Members in this RiPP family feature a thioamidated peptidyl chain conjugated with a macrocyclic ring system that contains two nonproteinogenic residues, 2-aminovinyl-cysteine (AviCys) and β-hydroxy-N,N-dimethyl-l-histidine (hdmHis). Focusing on the hdmHis residue that is unique to thioamitides, we report the enzymatic process for His functionalization and, more importantly, the timing of its related reactions with the other posttranslational modifications (PTMs) involved in thioamitide biosynthesis. His functionalization involves the activities of an S-adenosyl-l-methionine-dependent protein and a 2-oxoglutarate-Fe(II) monooxygenase for His bis-N-dimethylation and subsequent β-hydroxylation in a highly ordered manner. This process relies on the leader peptide sequence of the precursor peptide and on the establishment of the AviCys-containing, C-terminal macrocyclic ring system in particular. In contrast, prior peptide thioamidation is not required. Knowledge gained from the catalytic logic, specificity, and compatibility of His functionalization greatly furthers our understanding of the process through which nature develops thioamitides from a ribosomally synthesized peptide precursor.

Identification and characterization of a hemagglutinin gene from Prevotella intermedia

In this study, we describe the cloning of a gene encoding hemagglutinin from Prevotella intermedia to characterize hemagglutinin function and elucidate its possible role in the infectious process mediated by the organism. We screened genomic libraries created in Escherichia coli JM109, using antibodies against P . intermedia , and the positive clones were subsequently tested for their hemagglutinating activity, using rabbit red blood cells. We isolated a clone of strain 0543 in which, a 3.6-kb P. intermedia chromosomal DNA fragment was inserted into the pUC18 plasmid. Our deletion analysis-based subcloning procedures identified a hemagglutinin gene―designated phg. The open-reading frame of phg encoded a 35-kDa protein, and the expression of a recombinant protein of the predicted size was observed in a fusion protein expression system. While the DNA analysis of phg revealed no homology to known DNA sequences, the putative promoter, ribosome binding, and transcriptional termination sequence were found in the flanking DNA regions. In the N-terminus of the deduced amino acid sequence, we found a consensus leader peptide sequence for secretory proteins. These findings indicate that hemagglutinin might be expressed on the surface of P. intermedia to mediate its function.

Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors.

Clostridium perfringens is an opportunistic pathogenic bacterium that infects both animals and humans. Clostridium perfringens genomes encode a diverse array of toxins and virulence proteins, which continues to expand as more genomes are sequenced. In this study, the genomes of 44 C. perfringens strains isolated from intestinal sections of diseased cattle and from broiler chickens from diseased and healthy flocks were sequenced. These newly assembled genomes were compared to 141 publicly available C. perfringens genome assemblies, by aligning known toxin and virulence protein sequences in the assemblies using BLASTp. The genes for alpha toxin, collagenase, a sialidase (nanH), and alpha-clostripain were present in at least 99% of assemblies analyzed. In contrast, beta toxin, epsilon toxin, iota toxin, and binary enterotoxin of toxinotypes B, C, D, and E were present in less than 5% of assemblies analyzed. Additional sequence variants of beta2 toxin were detected, some of which were missing the leader or signal peptide sequences and therefore likely not secreted. Some pore-forming toxins involved in intestinal diseases were host-associated, the netB gene was only found in avian isolates, while netE, netF, and netG were only present in canine and equine isolates. Alveolysin was positively associated with canine and equine strains and only present in a single monophyletic clade. Strains from ruminant were not associated with known virulence factors and, except for the food poisoning associated clade, were present across the phylogenetic diversity identified to date for C. perfringens. Many C. perfringens strains associated with food poisoning lacked the genes for hyaluronidases and sialidases, important for attaching to and digesting complex carbohydrates found in animal tissues. Overall, the diversity of virulence factors in C. perfringens makes these species capable of causing disease in a wide variety of hosts and niches.

Importance of the leader peptide sequence on the lanthipeptide secretion level.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides. Their precursor peptide comprises of an N-terminal leader peptide and a C-terminal core peptide. Here, the leader peptide is crucial for enzyme recognition especially for the modification enzymes and acts furthermore as a secretion signal for the lanthipeptide exporter. The core peptide is the target site for the posttranslational modifications and contains dehydrated amino acids and lanthionine rings. Nisin produced by the Gram-positive bacterium Lactococcus lactis is one of the best-studied lanthipeptides and used as a model system to study their modification and secretion processes. Nisin is secreted as a precursor peptide. Here, we present an in vivo secretion analysis of NisT in the absence of the modification machinery allowing the secretion of leader peptide mutants and their impact solely on the secretion activity of NisT. Additionally, we created leader peptide hybrids to provide new insights, how the secretion is effected by unnatural leader peptides. The focus on the secretion activity of the transporter alone enabled us to determine the recognition site of NisT within the leader peptide of nisin.

Increased HLA-E Expression Correlates with Early Relapse in Multiple Myeloma

Increased HLA-E Expression Correlates with Early Relapse in Multiple Myeloma

Localization and activity of recombinant streptavidin in cell fractions of Escherichia coli

The authors provide a genetic construct for obtaining recombinant biologically active wild type streptavidin with high yield. Addition of leader peptide and oligohistidine sequence to the streptavidin sequence makes it possible to isolate soluble streptavidin from the culture medium and from the soluble fraction. Temperature conditions for inducing of protein synthesis were found to have a significant effect on the distribution profile of streptavidin between fractions. The obtained protein product is not contaminated with endogenous biotin. The provided method excludes denaturation and renaturation steps which are necessary for isolation of the desired product from inclusion bodies but substantially reduce the yield. Thus, the provided approach allows to increase the yield of biologically highly active strepatividin which is capable of binding biotin and biotin-containing compounds and can be used for various practical purposes.

NosP-Regulated Nosiheptide Production Responds to Both Peptidyl and Small-Molecule Ligands Derived from the Precursor Peptide

Nosiheptide, an archetypal member of thiopeptide antibiotics, arises from post-translational modifications of a ribosomally synthesized precursor peptidethat contains an N-terminal leader peptide (LP) sequence and a C-terminal core peptide (CP) sequence. Despite extensive efforts concerning the biosynthesis of thiopeptide antibiotics, the regulatory mechanisms in this process remain poorly understood. Using the nosiheptide-producing Streptomyces actuosus strain as a model system, we report here that NosP, a Streptomyces antibiotic regulatory protein, serves as the only cluster-situated regulator and activates the transcription of all structural genes, which are organized into two divergently transcribed operons in the nos cluster, by binding to their intergenic region. NocP, the counterpart of NosP in Nocardia sp., regulates the production of structurally related nocathiacin I in a similar manner. NosP activity senses the nosiheptide biosynthetic process by interactions with both peptidyl and small-molecule ligands that result from the LP and CP parts of the precursor peptide, respectively.

A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells

T-cell receptor (TCR) gene therapy is a promising approach for the treatment of infectious diseases and cancers. However, the paired cloning and functional assays of antigen-specific TCRα and TCRβ is time-consuming and laborious. In this study, we developed a novel, rapid and efficient antigen-specific TCR-cloning system by combining three technologies: multiplex one-step RT-PCR, transcriptionally active PCR (TAP) and luciferase reporter assays. Multiplex one-step RT-PCR with leader primers designed from leader peptide sequences of TCRs enabled us to amplify cDNAs of TCRα and β pairs from single T-cells with remarkably high efficiency. The combination of TAP fragments and HEK293T-based NFAT-luciferase reporter cells allowed for a rapid functional assay without the need to construct expression vectors. Using this system, we cloned human TCRs specific for Epstein-Barr virus BRLF-1-derived peptide as well as mouse TCRs specific for melanoma-associated antigen tyrosinase-related protein 2 (TRP-2) within four days. These results suggest that our system provides rapid and efficient cloning of functional antigen-specific human and mouse TCRs and contributes to TCR-based immunotherapy for cancers and infectious diseases.

Post-translational Claisen Condensation and Decarboxylation en Route to the Bicyclic Core of Pantocin A.

Pantocin A (PA) is a member of the growing family of ribosomally encoded and post-translationally modified peptide natural products (RiPPs). PA is much smaller than most known RiPPs, a tripeptide with a tight bicyclic core that appears to be cleaved from the middle of a larger 30-residue precursor peptide. We show here that the enzyme PaaA catalyzes the double dehydration and decarboxylation of two glutamic acid residues in the 30-residue precursor PaaP. Further truncates of PaaP leader and follower peptide sequences demonstrate the different impacts of these two regions on PaaA-mediated tailoring and delineate an essential role for the follower sequence in the decarboxylation step. The crystal structure of apo PaaA is reported, allowing identification of structural features that set PaaA apart from other homologous enzymes that typically do not catalyze such extended post-translational chemistry. Together, these data reveal how additional chemistry can be extracted from a ubiquitous enzyme family toward ribosomally derived peptide natural product biosynthesis and suggest that more examples of such enzymes likely exist in untapped genomic space.

Methanobactin and the Link between Copper and Bacterial Methane Oxidation.

Methanobactins (mbs) are low-molecular-mass (<1,200 Da) copper-binding peptides, or chalkophores, produced by many methane-oxidizing bacteria (methanotrophs). These molecules exhibit similarities to certain iron-binding siderophores but are expressed and secreted in response to copper limitation. Structurally, mbs are characterized by a pair of heterocyclic rings with associated thioamide groups that form the copper coordination site. One of the rings is always an oxazolone and the second ring an oxazolone, an imidazolone, or a pyrazinedione moiety. The mb molecule originates from a peptide precursor that undergoes a series of posttranslational modifications, including (i) ring formation, (ii) cleavage of a leader peptide sequence, and (iii) in some cases, addition of a sulfate group. Functionally, mbs represent the extracellular component of a copper acquisition system. Consistent with this role in copper acquisition, mbs have a high affinity for copper ions. Following binding, mbs rapidly reduce Cu(2+) to Cu(1+). In addition to binding copper, mbs will bind most transition metals and near-transition metals and protect the host methanotroph as well as other bacteria from toxic metals. Several other physiological functions have been assigned to mbs, based primarily on their redox and metal-binding properties. In this review, we examine the current state of knowledge of this novel type of metal-binding peptide. We also explore its potential applications, how mbs may alter the bioavailability of multiple metals, and the many roles mbs may play in the physiology of methanotrophs.