Non-canonical Peptide Research Articles

APE-Gen2.0: Expanding Rapid Class I Peptide-Major Histocompatibility Complex Modeling to Post-Translational Modifications and Noncanonical Peptide Geometries.

The recognition of peptides bound to class I major histocompatibility complex (MHC-I) receptors by T-cell receptors (TCRs) is a determinant of triggering the adaptive immune response. While the exact molecular features that drive the TCR recognition are still unknown, studies have suggested that the geometry of the joint peptide-MHC (pMHC) structure plays an important role. As such, there is a definite need for methods and tools that accurately predict the structure of the peptide bound to the MHC-I receptor. In the past few years, many pMHC structural modeling tools have emerged that provide high-quality modeled structures in the general case. However, there are numerous instances of non-canonical cases in the immunopeptidome that the majority of pMHC modeling tools do not attend to, most notably, peptides that exhibit non-standard amino acids and post-translational modifications (PTMs) or peptides that assume non-canonical geometries in the MHC binding cleft. Such chemical and structural properties have been shown to be present in neoantigens; therefore, accurate structural modeling of these instances can be vital for cancer immunotherapy. To this end, we have developed APE-Gen2.0, a tool that improves upon its predecessor and other pMHC modeling tools, both in terms of modeling accuracy and the available modeling range of non-canonical peptide cases. Some of the improvements include (i) the ability to model peptides that have different types of PTMs such as phosphorylation, nitration, and citrullination; (ii) a new and improved anchor identification routine in order to identify and model peptides that exhibit a non-canonical anchor conformation; and (iii) a web server that provides a platform for easy and accessible pMHC modeling. We further show that structures predicted by APE-Gen2.0 can be used to assess the effects that PTMs have in binding affinity in a more accurate manner than just using solely the sequence of the peptide. APE-Gen2.0 is freely available at https://apegen.kavrakilab.org.

MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

Abstract A038: Non-canonical MHC class I-associated antigens in pancreatic cancer

Abstract Translation of the ostensibly non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I, HLA-I in humans). These non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies, typified by pancreatic ductal adenocarcinoma (PDA). However, non-canonical MHC-I-associated peptides (ncMAPs) have yet to be explored in pancreatic cancer. We purified the malignant compartment from low tumor cellularity PDA specimens using patient-derived organoids (PDOs) and developed a personalized proteogenomics pipeline coupled with high-depth immunopeptidomics to deeply characterize the repertoire of HLA:peptide targets presented specifically on malignant cells. We demonstrate that ncMAPs are abundant on PDA and predominate over mutation-derived neoepitopes in the detectable immunopeptidome. While emerging evidence in other tumor types has implicated dysregulated translation of non-canonical open reading frames (ncORFs), it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting ncMAPs can elicit a cytolytic T lymphocyte (CTL) response. To investigate the cancer-specificity of non-canonical peptides, we developed a translation-centric analysis pipeline that examines ncORF expression across a range of healthy tissues, including healthy thymus. We provide evidence that ~30% of ncMAPs exhibit cancer-restricted translation patterns, and a substantial subset of these are shared among pancreatic cancer patients with the appropriate HLA. To investigate immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand antigen-specific T cells. We demonstrate that PDA-restricted ncMAPs are highly immunogenic, on par with or even exceeding the immunogenicity observed with mutation-derived peptides or tumor-associated antigens assayed using the same platform. These findings uncover a critical role for dysregulated translation in pancreatic cancer as a potential source for recurrent cancer-restricted epitopes capable of recognition by cytotoxic T cells. We envision that this novel class of antigens will accelerate ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies. Citation Format: Zackery A. Ely, Zachary J. Kulstad, Eva K. Verzani, Jennifer G. Abelin, Sudarsana Addepalli, Karl R. Clauser, Marta Casarrubios, Kevin S. Kapner, Miles P. Agus, Connor J. Hennessey, Sine R. Hadrup, Susan Klaeger, Jennifer Su, Alex M. Jaeger, Brian M. Wolpin, Srivatsan Raghavan, Philip D. Greenberg, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks, William A. Freed-Pastor. Non-canonical MHC class I-associated antigens in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A038.

Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H+/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. KM and relative Vmax values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances.

In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease

γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.

A Post-Synthetic Modification Strategy for the Preparation of Homooligomers of 3-Amino-1-methylazetidine-3-carboxylic Acid

AbstractPost-synthetic modification is a powerful technique allowing access to noncanonical peptide derivatives in a selective manner, but it has not so far been applied for the installation of multiple arrays of modified side chains. Here, we use this approach in solution phase to prepare short N- and C-capped homooligomers of 3-amino-1-methylazetidine-3-carboxylic acid with all the azetidine side chain functions in free amine form. The key step is the multiple reductive amination reaction of the corresponding post-synthetically deprotected secondary amines.

Abstract C014: Broadening the repertoire of PDAC-specific targets for immune-based therapy through high-resolution immunopeptidomics

Abstract Pancreatic adenocarcinoma (PDAC) is among the most lethal cancer types and has been largely recalcitrant to traditional immunotherapy. A large subset of PDAC tumors is computationally predicted to harbor potentially immunogenic peptides for MHC class I (MHC-I) presentation, but the nature, expression, and immunogenicity of these peptides has yet to be determined. By investigating the PDAC immunopeptidome, we can uncover and exploit novel immune-based targets for PDAC and render it vulnerable to immunotherapy. Prior efforts to study the immunopeptidome in PDAC have largely focused on profiling MHC-associated peptides (MAPs) from bulk tumor samples. This approach is severely limited by the contribution of MAPs from the non-malignant compartments, which constitutes most of the cellular mass in PDAC. We can overcome this limitation by using patient-derived organoids (PDOs) to expand a pure cancer cell population for MHC-I immunoprecipitation, followed by LC/MS-MS. We applied this approach and detected 17,000-20,500 unique MAPs per sample, a dramatic increase in depth and resolution over prior efforts. To ascertain which MAPs may be PDAC-restricted, we first analyzed bulk RNA-sequencing data from the Genotype-Tissue Expression Project (767 patients, 30 tissues) to generate a set of genes that are functionally undetectable (Q90 <1 TPM) in healthy somatic tissues. We cross-referenced this list with our set of PDO MAPs, yielding 143 PDAC-restricted MAPs. To further expand our search space, we implemented a tiered tissue-based filter to relax TPM cutoffs in less essential tissues (ex. prostate) while maintaining strict cutoffs in essential tissues (ex. brain), generating 85 additional MAPs. Both approaches uncovered cancer-restricted MAPs present in most PDO lines, which may represent shared therapeutic targets. While somatic mutations are a well-established source of tumor-specific neoantigens, these have yet to be investigated with immunopeptidomics in human PDAC. Despite harboring an intermediate mutational burden, we detected at least one mutation-derived neoepitope in most PDOs, a detection frequency much closer to the detection frequency of cancers with high mutational burden than previously expected. Additionally, non-canonical peptide sources, including retained introns (RI) and novel unannotated open reading frames (nuORF), may represent a source of cancer-restricted MAPs. Interestingly, we detect nuORF- and RI-derived MAPs (n=298) in all PDOs, suggesting that these too may be therapeutically relevant in PDAC. To assess the immunogenicity of candidate MAPs, future studies will utilize established protocols for priming and expanding MAP-specific human T cells with autologous DCs. Functional evaluation of MAP-specific T cells will help prioritize strategies for vaccination, and the generation of T cell receptor sequences for adoptive cell therapy. Collectively, these data deepen our understanding of the PDAC immunopeptidome and provide a novel set of targets for immunotherapy in PDAC. Citation Format: Zackery A. Ely, William A. Freed-Pastor, Zachary J. Kulstad, Jennifer G. Abelin, Eva Verzani, Kevin S. Kapner, Susan Klaeger, Karl R. Clauser, Miles Agus, Alex M. Jaeger, Nimisha B. Pattada, Arjun Bhutkar, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks. Broadening the repertoire of PDAC-specific targets for immune-based therapy through high-resolution immunopeptidomics [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C014.

Late-Stage Modification of Oligopeptides by Nickel-Catalyzed Stereoselective Radical Addition to Dehydroalanine.

Radical addition to dehydroalanine (Dha) represents an appealing, modular strategy to access non-canonical peptide analogues for drug discovery. Prior studies on radical addition to the Dha residue of peptides and proteins have demonstrated outstanding functional group compatibility, but the lack of stereoselectivity has limited the synthetic utility of this approach. Herein, we address this challenge by employing chiral nickel catalysts to control the stereoselectivity of radical addition to Dha on oligopeptides. The conditions accommodate a variety of primary and secondary electrophiles to introduce polyethylene glycol, biotin, halo-tag, and hydrophobic and hydrophilic side chains to the peptide. The reaction features catalyst control to largely override substrate-based control of stereochemical outcome for modification of short peptides. We anticipate that the discovery of chiral nickel complexes that confer catalyst control will allow rapid, late-stage modification of peptides featuring nonnatural sidechains.

InSPIRE: An Open-Source Tool for Increased Mass Spectrometry Identification Rates Using Prosit Spectral Prediction

Rescoring of mass spectrometry (MS) search results using spectral predictors can strongly increase peptide spectrum match (PSM) identification rates. This approach is particularly effective when aiming to search MS data against large databases, for example, when dealing with nonspecific cleavage in immunopeptidomics or inflation of the reference database for noncanonical peptide identification. Here, we present inSPIRE (in silico Spectral Predictor Informed REscoring), a flexible and performant open-source rescoring pipeline built on Prosit MS spectral prediction, which is compatible with common database search engines. inSPIRE allows large-scale rescoring with data from multiple MS search files, increases sensitivity to minor differences in amino acid residue position, and can be applied to various MS sample types, including tryptic proteome digestions and immunopeptidomes. inSPIRE boosts PSM identification rates in immunopeptidomics, leading to better performance than the original Prosit rescoring pipeline, as confirmed by benchmarking of inSPIRE performance on ground truth datasets. The integration of various features in the inSPIRE backbone further boosts the PSM identification in immunopeptidomics, with a potential benefit for the identification of noncanonical peptides.

Quantitative Proteogenomic Characterization of Inflamed Murine Colon Tissue Using an Integrated Discovery, Verification, and Validation Proteogenomic Workflow.

Chronic inflammation of the colon causes genomic and/or transcriptomic events, which can lead to expression of non-canonical protein sequences contributing to oncogenesis. To better understand these mechanisms, Rag2−/−Il10−/− mice were infected with Helicobacter hepaticus to induce chronic inflammation of the cecum and the colon. Transcriptomic data from harvested proximal colon samples were used to generate a customized FASTA database containing non-canonical protein sequences. Using a proteogenomic approach, mass spectrometry data for proximal colon proteins were searched against this custom FASTA database using the Galaxy for Proteomics (Galaxy-P) platform. In addition to the increased abundance in inflammatory response proteins, we also discovered several non-canonical peptide sequences derived from unique proteoforms. We confirmed the veracity of these novel sequences using an automated bioinformatics verification workflow with targeted MS-based assays for peptide validation. Our bioinformatics discovery workflow identified 235 putative non-canonical peptide sequences, of which 58 were verified with high confidence and 39 were validated in targeted proteomics assays. This study provides insights into challenges faced when identifying non-canonical peptides using a proteogenomics approach and demonstrates an integrated workflow addressing these challenges. Our bioinformatic discovery and verification workflow is publicly available and accessible via the Galaxy platform and should be valuable in non-canonical peptide identification using proteogenomics.

Rapid de novo discovery of peptidomimetic affinity reagents for human angiotensin converting enzyme 2

Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging. Angiotensin converting enzyme 2 (ACE2) has recently been identified as a cardiovascular disease biomarker and the primary receptor utilized by the severe acute respiratory syndrome coronavirus 2. In this study, we report the discovery of high affinity peptidomimetic binders to ACE2 via affinity selection-mass spectrometry (AS-MS). Multiple high affinity ACE2-binding peptides (ABP) were identified by selection from canonical and noncanonical peptidomimetic libraries containing 200 million members (dissociation constant, KD = 19–123 nM). The most potent noncanonical ACE2 peptide binder, ABP N1 (KD = 19 nM), showed enhanced serum stability in comparison with the most potent canonical binder, ABP C7 (KD = 26 nM). Picomolar to low nanomolar ACE2 concentrations in human serum were detected selectively using ABP N1 in an enzyme-linked immunosorbent assay. The discovery of serum-stable noncanonical peptidomimetics like ABP N1 from a single-pass selection demonstrates the utility of advanced AS-MS for accelerated development of affinity reagents to protein targets.

Enzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase.

Nonribosomal peptide synthetases (NRPSs) are typically multimodular enzymes that assemble amino acids or carboxylic acids into complex natural products. Here, we characterize a monomodular NRPS, PvfC, encoded by the Pseudomonas virulence factor (pvf) gene cluster that is essential for virulence and signaling in different bacterial species. PvfC exhibits a unique adenylation-thiolation-reductase (ATR) domain architecture that is understudied in bacteria. We show that the activity of PvfC is essential in the production of seven leucine-derived heterocyclic natural products, including two pyrazines, a pyrazinone, and a rare disubstituted imidazole, as well as three pyrazine N-oxides that require an additional N-oxygenation step. Mechanistic studies reveal that PvfC, without a canonical peptide-forming domain, makes a dipeptide aldehyde intermediate en route to both the pyrazinone and imidazole. Our work identifies a novel biosynthetic route for the production of pyrazinones, an emerging class of signaling molecules and virulence factors. Our discovery also showcases the ability of monomodular NRPSs to generate amino acid- and dipeptide-aldehydes that lead to diverse natural products. The diversity-prone biosynthesis by the pvf-encoded enzymes sets the stage for further understanding the functions of pvf in bacterial cell-to-cell signaling.

Fluorometric Optimized Determination of Total Glutathione in Erythrocytes

Glutathione is a tripeptide natural product characterized by a non-canonical peptide bond with an amide moiety linking the nitrogen of cysteine to the γ-carboxyl of glutamate, and is found ubiquitously in nature, in animals, plants and microorganisms. One of the most abundant biological matrices is represented by erythrocytes, being glutathione the only sulfur-containing mechanism for the red blood cell oxidative protection. Several analytical methods for glutathione determination from different samples are described in the literature and most of these methods are based on the use of high-performance liquid chromatography. HPLC equipment is not available in all the biochemical laboratories, and, moreover, displays lot of economic and ecological limitations, including organic solvent consumption and time-consuming analysis. Here, an organic-free high-throughput fluorometric methodology for the analysis of total glutathione in erythrocytes is reported, avoiding the use of time-consuming and not-sustainable techniques.

Synthesis of Non-canonical Amino Acids and Peptide Containing Them for Establishment of the Template for Drug Discovery.

Non-canonical amino acid derivatives are an attractive scaffold for novel drug candidates. Among the methods used to prepare this motif, the asymmetric Mannich-type reaction of α-imino carboxylic acid derivatives is a preeminent strategy because a wide variety of non-canonical amino acids can be accessed by changing only the nucleophile. Preparing the common substrate is difficult, however, which makes this method problematic. We developed a convenient method for synthesizing common substrates using MnO2-mediated oxidation of stable precursors. Peptides bearing non-canonical amino acids are another attractive synthetic target. We propose a new approach for synthesizing non-canonical amino acid-containing peptides by directly applying various organic reactions to peptidic substrates. Using hydrophobic anchor-supported peptides, we directly applied ring-closing metathesis and asymmetric Friedel-Crafts reactions to peptidic substrates. We also developed a novel recyclable organocatalyst according to the nature of the hydrophobic anchor tagged compound.

Enzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase

Enzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase

Proteogenomic Analysis Unveils the HLA Class I Presented Immunopeptidome in Melanoma and EGFR Mutant Lung Adenocarcinoma

Immune checkpoint inhibitor and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential for cancers with high tumor mutation burden (TMB). Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class ǀ-presented peptides in both melanoma, a “hot tumor” with high TMB, and EGFR mutant lung adenocarcinoma, a “cold tumor” with low TMB. We identified several classes of neopeptides, including mutated neoantigens and more than 1000 post-translationally modified peptides representing 58 different PTMs. We constructed a cancer germline (CG) antigen database with 285 antigens and identified 42 Class I-presented CG antigens. Finally, we developed a non-canonical peptide discovery pipeline to identify 44 lncRNA-derived peptides and validated Class I binding for select neopeptides. We provide direct evidence of HLA Class I presentation of a large number of neopeptides for potential vaccine or adoptive cell therapy in melanoma and mutant EGFR lung cancer.

Biochemical characterization of the Nocardia lactamduransACV synthetase.

The L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS) is a nonribosomal peptide synthetase (NRPS) that fulfills a crucial role in the synthesis of β-lactams. Although some of the enzymological aspects of this enzyme have been elucidated, its large size, at over 400 kDa, has hampered heterologous expression and stable purification attempts. Here we have successfully overexpressed the Nocardia lactamdurans ACVS in E. coli HM0079. The protein was purified to homogeneity and characterized for tripeptide formation with a focus on the substrate specificity of the three modules. The first L-α-aminoadipic acid-activating module is highly specific, whereas the modules for L-cysteine and L-valine are more promiscuous. Engineering of the first module of ACVS confirmed the strict specificity observed towards its substrate, which can be understood in terms of the non-canonical peptide bond position.

Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.

Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine.

Enzymatically crosslinked microporous hydrogel scaffolds that form in situ promote dermal healing Evaluation of: Griffin DR, Weaver WM, Scumpia PO, DiCarlo D, Segura T. Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat. Mater. 14, 737–744 (2015). Tunable scaffolds that respond to environmental factors could enable enhanced tissue integration and wound healing. To achieve this goal, scaffold designs have incorporated biomimetic functionality, including thrombin-activated factor XIII (FXIIIa) crosslinking [1], matrix metalloproteinase responsiveness [2] and mechanical matching [3]. Scaffolds that can alter their properties in situ in response to environmental stimuli are desired to achieve changes in stiffness, shape, release and degradation that can lead to improved tissue regeneration. In this study, Griffin et al. describe a bottom-up strategy to fabricating scaffolds that accelerate wound healing [4]. They use a microfluidic approach to form hydrogel microparticle (microgel) building blocks that can establish interconnected network structures upon FXIIIa-mediated crosslinking. These microporous annealed particles (MAPs) incorporate reactive multiarmed poly(ethylene)glycol-vinyl sulfone, the commonly used RGD cell adhesion peptide and the K and Q noncanonical transglutaminase peptide substrates with cysteine-terminated matrix metalloproteinase-sensitive peptides that allow for cell-controlled material degradation. Scaffolds are annealed by FXIIIa addition and can be injected into wound sites for in situ solidification and shape-conformation. These MAPs are mechanically robust and can be tuned to achieve storage moduli from 10 to 1000 Pa, which fall within the stiffness range of soft tissues. In vitro studies demonstrate that MAP scaffolds enable 3D cellular network formation and cell proliferation and survival after 6 days. In vivo studies in SKH1-HR and Balb/c epidermal wound healing models demonstrate integration of MAP scaffolds after 24 h, significant wound closure as compared with nonporous chemically matched controls after 5 days or physically matched control scaffolds up to 7 days. 5 days following injection of MAP scaffolds, keratin-5, keratin-14 and CD49f staining revealed re-epithelialization, and PECAM-1, NG2 and PDGFR-β staining indicated that the scaffolds promoted complex vascular development. MAP scaffolds were also able to sustain normal hair follicle formation when compared with normal tissue. The scaffolds promoted less CD11b cell infiltration as compared with nonporous controls after 5 days, which suggests that MAP scaffolds cause a lower immune response. Taken together, MAP scaffolds promote accelerated wound healing by utilizing natural enzymatic mechanisms and mimicking favorable mechanical and degradation properties to facilitate cell infiltration and proliferation. The study may have benefited from evaluating wound closure, dermal regeneration and Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis.

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.