Peptide Backbone Research Articles

α-Methylation Enables the X-ray Crystallographic Observation of Oligomeric Assemblies Formed by a β-Hairpin Peptide Derived from Aβ.

The assembly of the β-amyloid peptide Aβ into toxic oligomers plays a significant role in the neurodegeneration associated with the pathogenesis of Alzheimer's disease. Our laboratory has developed N-methylation as a tool to enable X-ray crystallographic studies of oligomers formed by macrocyclic β-hairpin peptides derived from Aβ. In this investigation, we set out to determine whether α-methylation could be used as an alternative to N-methylation in studying the oligomerization of a β-hairpin peptide derived from Aβ. α-Methylation permits the crystallographic assembly of a triangular trimer and ball-shaped dodecamer, resembling assemblies formed by the N-methylated homolog. Subtle differences are observed in the conformation of the α-methylated peptide when compared to the N-methylated homolog. Notably, α-methylation appears to promote a flatter and more extended β-sheet conformation than that of N-methylated β-sheets or a typical unmodified β-sheet. α-Methylation provides an alternative to N-methylation in X-ray crystallographic studies of oligomers formed by peptides derived from Aβ, with the attractive feature of preserving NH hydrogen-bond donors along the peptide backbone.

Effect of a single water molecule on the conformational preferences of a capped Pro–Gly dipeptide in the gas phase

Herein, we have investigated the effect of microhydration on the secondary structure of a capped dipeptide Boc-DPro-Gly-NHBn-OMe (Boc = tert-butyloxycarbonyl, Bn = Benzyl), i.e., Pro–Gly (PG) with a single H2O molecule using gas-phase laser spectroscopy combined with quantum chemistry calculations. Observation of a single conformer of the monohydrated peptide has been confirmed from IR-UV hole-burning spectroscopy. Both gas-phase experimental and theoretical IR spectroscopy results confirm that the H2O molecule is inserted selectively into the relatively weak C7 hydrogen bond (γ-turn) between the Pro C=O and NHBn N–H groups of the peptide, while the other C7 hydrogen bond (γ-turn) between the Gly N–H and Boc C=O groups remains unaffected. Hence, the single H2O molecule in the PG⋯(H2O)1 complex significantly distorts the peptide backbone without appreciable modification of the overall secondary structural motif (γ–γ) of the isolated PG monomer. The nature and strength of the intra- and inter-molecular hydrogen bonds present in the assigned conformer of the PG⋯(H2O)1 complex has also been examined by natural bond orbital and non-covalent interaction analyses. The present investigation on the monohydrated peptide demonstrates that several H2O molecules may be required for switching the secondary structure of PG from the double γ-turn to a β-turn that is favorable in the condensed phase.

Site-Specific N-Glycoprofiling of Immunoglobulin G Subtypes from Donkey Milk.

Donkey milk IgG was probed for the site-specific N-glycosylation pattern through RP-UHPLC-MS/MS. The affinity-purified milk IgG was subjected to SDS-PAGE and proteomic analysis, which revealed the presence of subtypes. Multiple N-glycopeptides arising from the predicted donkey IgG1, IgG2, IgG3, IgG5, IgG6, and IgG7 subtypes' heavy-chain constant region were shown to contain glycans at the highly conserved glycosylation site NST in the CH2 domain. Differences in the peptide backbone with the NST site among subtypes generated after trypsin digestion resulted in the evaluation of the subtype-specific glycan pattern. Glycan sequence analysis indicated predominantly biantennary complex types with core fucosylation at the site NST. Interestingly, an additional site NQT in the CH1 domain of the heavy-chain constant region of IgG5 was found to possess mainly sialylated biantennary complex glycans with NeuAc and NeuGc. Structural diversity of glycans was mainly observed in the predicted donkey IgG1, IgG5, and IgG7, whereas IgG2, IgG3, and IgG6 resulted in the glycopeptides that are of low abundance in the analyzed samples. These findings would pave the way for a better understanding of donkey milk functional properties.

End Group Effects on Anion Binding in Tetraglycine Peptide: A Computational Study.

The importance of anions in various processes has led to a search for molecules that can effectively recognize and interact with these anions. This study explores how the tetraglycine [(Gly)4] peptide in its zwitterionic, neutral, and terminally capped forms acts as a receptor for H2PO4 - and HSO4 - anions within the framework of supramolecular host-guest chemistry. Using molecular dynamics (MD) simulations, we obtained the conformations of the receptor-anion complexes. Density functional theory (DFT), quantifies the complexes' interaction energies in both gas and solvent phases. Proton transfer within the zwitterionic complex with H2PO4 - anion alters peptide charge distribution, affecting its conformation and binding site arrangement, as analysed by quantum mechanics/molecular mechanics (QM/MM) methods. Symmetry-adapted perturbation theory (SAPT) and noncovalent interactions analysis highlight the role of electrostatic interactions in these receptor-anion complexes. It emphasizes the key interactions such as N-H⋅⋅⋅⋅O and O-H⋅⋅⋅O=C between the peptide backbone and anions and elucidates the molecular recognition mechanism driven by crucial noncovalent interactions. The termination of the peptide's end groups modulates anion binding sites from the backbone to the charged N-terminal, resulting in distinct binding sites. Our findings provide insights for designing peptides tailored to function as anion receptors in diverse supramolecular chemistry applications.

Unravelling the Structure of One-Electron Oxidation Products of Model Peptide Backbones Containing Methionine by IRMPD Spectroscopy: the Effect of the Neighbouring Groups.

A series of Methionine (Met) derivatives, where either the amino group and/or the carboxylic acid group is blocked by acetyl and/or methyl ester functionalities, has been investigated by Collision Induced Dissociation-tandem mass spectrometry (CID-MS2) and Infrared Multiple Photon Dissociation (IRMPD) spectroscopy. The CID-MS2 experiments were performed using a Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometer equipped with an electrospray ionization (ESI) source. The IRMPD spectra were recorded employing a Paul type ion-trap coupled with the free-electron laser (FEL) FELIX in the fingerprint region from 600 to 2000 cm-1. We show that the oxidation of the methionine residue with protected terminal groups induces the formation of a sulfoxide function. However, compared to the IRMPD spectrum of protonated methionine and methionine sulfoxide, significant spectral differences are observed in the spectra of model protected peptide backbones containing methionine. DFT calculations show that protonation occurs on the sulfoxide group in the gas phase for these model peptide backbones containing methionine, shifting the diagnostic signature of the sulfoxide group from 1000 to 862 cm-1.

Non‐Natural MUC1 Glycopeptide Homogeneous Cancer Vaccine with Enhanced Immunogenicity and Therapeutic Activity

AbstractGlycopeptides derived from the glycoprotein mucin‐1 (MUC1) have shown potential as tumor‐associated antigens for cancer vaccine development. However, their low immunogenicity and non‐selective conjugation to carriers present significant challenges for the clinical efficacy of MUC1‐based vaccines. Here, we introduce a novel vaccine candidate based on a structure‐guided design of an artificial antigen derived from MUC1 glycopeptide. This engineered antigen contains two non‐natural amino acids and has an α‐S‐glycosidic bond, where sulfur replaces the conventional oxygen atom linking the peptide backbone to the sugar N‐acetylgalactosamine. The glycopeptide is then specifically conjugated to the immunogenic protein carrier CRM197 (Cross‐Reactive Material 197), a protein approved for human use. Conjugation involves selective reduction and re‐bridging of a disulfide in CRM197, allowing the attachment of a single copy of MUC1. This strategy results in a chemically defined vaccine while maintaining both the structural integrity and immunogenicity of the protein carrier. The vaccine elicits a robust Th1‐like immune response in mice and generates antibodies capable of recognizing human cancer cells expressing tumor‐associated MUC1. When tested in mouse models of colon adenocarcinoma and pancreatic cancer, the vaccine is effective both as a prophylactic and therapeutic use, significantly delaying tumor growth. In therapeutic applications, improved outcomes were observed when the vaccine was combined with an anti‐programmed cell death protein 1 (anti‐PD‐1) checkpoint inhibitor. Our strategy reduces batch‐to‐batch variability and enhances both immunogenicity and therapeutic potential. This site‐specific approach disputes a prevailing dogma where glycoconjugate vaccines require multivalent display of antigens.

Peptide core spherical nucleic acids circumvent tumor immunosuppression via supplementing methionine for enhanced photodynamic/gene immune/therapy of hepatocellular carcinoma

Spherical nucleic acids (SNAs) with functional peptide cores are an emerging nanoplatform for synergistic cancer therapy but have been rarely reported. We construct herein the first SNA nanoplatform based on a biodegradable binary peptide backbone of methionine (Met) and cysteine (Cys) for codelivery of a photosensitizer, Chlorin e6 (Ce6) and human liver-specific miR122 for synergistic photodynamic-gene therapy of hepatic cell carcinoma (HCC). Met supplementation by the peptide core improves the infiltration of T cells and enhances the effector function of T cells for turning a “cold” tumor into a “hot” one. The resulting SNA(+) shows the most significant inhibitory effect in a Hepa1-6 HCC primary/distal tumor model, with tumor growth inhibition (TGI) values of 98.5 ± 0.5 % and 99.1 ± 0.4 % for the primary and distant tumors, respectively. This SNA nanoplatform achieves superior high TGI values reported thus far to our knowledge with almost complete eradication for both tumors due to the simultaneous adaptive and innate immunity activation via photodynamic therapy (PDT) induced immunogenic cell death (ICD) and Met supplementation-promoted adaptive immunity, and miR122-enhanced innate immunity. Overall, this study not only develops a reliable synthetic strategy toward peptide-backboned multifunctional SNA nanoplatform, but also reports the modulation of amino acid metabolism for enhanced innate immunity for highly efficient HCC immunotherapy.

Profiling Cysteine Proteases Activities in Neuroinflammatory Cells.

A new activity-based probe (ABP) of cysteine proteases (FGA139) has been designed and synthesized. The design of the ABP has been done based upon the chemical structure of an irreversible inhibitor of cysteine proteases by attaching a bodipy fluorophore at the N-terminus of the peptide backbone. The synthetic route of the probe has a metathesis and a "click" reaction as key steps. Although some studies have been reported about the role played by cysteine proteases in neurodegenerative diseases, there are not definitive conclusions. The obtained ABP has been employed as a chemical tool to profile activities of cysteine proteases cathepsins B, L, and calpain in neurodegenerative cell models through confocal imaging. Colocalization of the probe to specific antibodies of the proteases and competitive experiments with non-fluorescent inhibitors confirm the specificity of the ABP. From a theranostic perspective, our findings strongly suggest that FGA139 exhibits a protective role in various cell lines against oxidative stress or pro-inflammatory toxicity and it effectively attenuates macrophage activation triggered by LPS.

Unravelling the favorability of radical-directed xn-H2O dissociation at serine and threonine

Among tandem-mass-spectrometry approaches, radical-directed dissociation (RDD) is uniquely sensitive to molecular structure because the location and types of cleavage observed are dictated by radical migration propensities. Although the underlying chemistry for many RDD fragmentation pathways has been previously explained, xn-H2O fragment ions that occur exclusively at serine and threonine residues, have not been examined in detail. Creation of this fragment type inherently requires two dissociation events, one to lose water and another to cleave the peptide backbone. Double dissociations are typically disfavored relative to pathways requiring a single cleavage, yet xn-H2O fragment ions are abundant in RDD spectra. To understand why this fragmentation pathway is favorable, we used a combination of computational chemistry and experiments on peptides with a variety of covalent modifications. Our results explore the energetics, location, and migration of the radical in each step of the mechanism, revealing that favorability can be attributed to the stability of the required radical intermediates and access to low-energy pathways connecting them. Ultimately, the abundant nature of xn-H2O ions and the selectivity associated with their exclusive generation at Ser/Thr provides high value sequence information in RDD experiments.

Efficiency Enhancement in Peptide Hydrogelators: The Crucial Role of Side Chain Hydrogen Bonding Over Aromatic π-π Interactions.

Short peptide assemblies that form supramolecular hydrogels are stabilized by both intermolecular noncovalent interactions among amino acid side chains and hydrogen bonding between peptide backbone amides. Previous research has emphasized the inclusion of aromatic amino acids in short peptide sequences, positing that aromatic π-π interactions contribute significantly to inducing efficient hydrogelation i.e., at low minimum gelation concentrations (MGCs). However, herein, we demonstrate that additional hydrogen bonding interactions from amino acid side chains play a more pivotal role in the efficiency of peptide hydrogelation compared to aromatic π-π interactions. We investigated two sets of Fluorenylmethoxycarbonyl (Fmoc)-functionalized α-synuclein and human islet amyloid peptide fragments [Fmoc-NVGGAVVT (Syn-N) and Fmoc-NFGAIL (IAP-N)], substituting asparagine (N) with phenylalanine (Syn-F/IAP-F), alanine (Syn-A/IAP-A), and glutamine (Syn-Q/IAP-Q). This allowed us to explore the effects of aromatic (π-system), aliphatic (hydrophobic), and hydrogen bonding effects with varying chain lengths on hydrogel formation. Our results reveal that Syn-N and Syn-Q exhibit MGC of 0.03 and 0.05 wt %, respectively, classifying them as super hydrogelators (MGC < 0.1 wt %). These values are 4.0-6.6-fold lower than Syn-F and Syn-A, with Syn-N demonstrating greater efficiency than Syn-Q. Similarly, IAP-N exhibited a substantial decrease in MGC by 8.75, 3.75, and 2.5 folds compared to IAP-A, IAP-F, and IAP-Q, respectively. Experimental evidence and molecular dynamic simulation suggest that -CO-NH2 of asparagine side chains effectively engaged in hydrogen bonding, thereby immobilizing water molecules at low gelator concentrations. Although glutamine shares similar -CO-NH2 functionality, its hydrogelation efficiency is less pronounced compared to asparagine, likely due to its longer alkyl chain, which may hinder the formation of a hydrogen bonding network in the self-assembled structure compared to asparagine-containing peptides. These findings offer valuable insights for designing efficient peptide hydrogelators or lowering MGCs by substituting amino acids with asparagine/glutamine in peptide sequences. Additionally, modifying peptide properties through asparagine/glutamine substitution could optimize hydrogel properties for specific applications.

Phosphorylation of the HMGN1 Nucleosome Binding Domain Decreases Helicity and Interactions with the Acidic Patch.

Intrinsically disordered proteins are abundant in the nucleus and are prime sites for posttranslational modifications that modulate transcriptional regulation. Lacking a defined three-dimensional structure, intrinsically disordered proteins populate an ensemble of several conformational states, which are dynamic and often altered by posttranslational modifications, or by binding to interaction partners. Although there is growing appreciation for the role that intrinsically disordered regions have in regulating protein-protein interactions, we still have a poor understanding of how to determine conformational population shifts, their causes under various conditions, and how to represent and model conformational ensembles. Here, we study the effects of serine phosphorylation in the nucleosome-binding domain of an intrinsically disordered protein - HMGN1 - using NMR spectroscopy, circular dichroism and modelling of protein complexes. We show that phosphorylation induces local conformational changes in the peptide backbone and decreases the helical propensity of the nucleosome binding domain. Modelling studies using AlphaFold3 suggest that phosphorylation disrupts the interface between HMGN1 and the nucleosome acidic patch, but that the models over-predict helicity in comparison to experimental data. These studies help us to build a picture of how posttranslational modifications might shift the conformational populations of disordered regions, alter access to histones, and regulate chromatin compaction.

Exploring novel antioxidant cyclic peptides in corn protein hydrolysate: Preparation, identification and molecular docking analysis

Antioxidant cyclic peptides were successfully identified from a corn protein hydrolysate. Hydrolysate by Alcalase + Flavourzyme showed the highest cyclic peptide purity (48.36 ± 1.81 %) and higher antioxidant activities compared with other hydrolysate. The success of peptide cyclization in hydrolysate was demonstrated by thermogravimetric analysis and thin-layer chromatography (TLC) analysis. Thermogravimetric analysis showed that the thermal stability of hydrolysate after cyclization was significantly increased, which was related to the formation of cyclic peptides. Peptides with molecular weight less than 1000 Da accounted for more than 80 % in hydrolysate after cyclization. After separation using gel silica chromatography and semi-preparative reverse phase high performance liquid chromatography (RP-HPLC), 22 novel antioxidant cyclic peptides were identified by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) and orbitrap-tandem mass spectrometry (Orbitrap-MS/MS). Synthetic cyclic peptides with the same sequence were synthesized and characterized for their antioxidant activity. Molecular docking suggested that the free radical molecules could bind with the cyclic backbone and side chain of cyclic peptides through hydrogen bonding, hydrophobic interaction as well as electrostatic interaction. This study has important implications for the high-value utilization of corn protein and new cyclic peptides drugs or functional food development.

Potent and Protease Resistant Azapeptide Agonists of the GLP‐1 and GIP Receptors

AbstractThe gut‐derived peptide hormones glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) play important physiological roles including glucose homeostasis and appetite suppression. Stabilized agonists of the GLP‐1 receptor (GLP‐1R) and dual agonists of GLP‐1R and GIP receptor (GIPR) for the management of type 2 diabetes and obesity have generated widespread enthusiasm and have become blockbuster drugs. These therapeutics are refractory to the action of dipeptidyl peptidase‐4 (DPP4), that catalyzes rapid removal of the two N‐terminal residues of the native peptides, in turn severely diminishing their activity profiles. Here we report that a single atom change from carbon to nitrogen in the backbone of the entire peptide makes them refractory to DPP4 action while still retaining full potency and efficacy at their respective receptors. This was accomplished by use of aza‐amino acids, that are bioisosteric replacements for α‐amino acids that perturb the structural backbone and local side chain conformations. Molecular dynamics simulations reveal that aza‐amino acid can populate the same conformational space that GLP‐1 adopts when bound to the GLP‐1R. The insertion of an aza‐amino acid at the second position from the N‐terminus in semaglutide and in a dual agonist of GLP‐1R and GIPR further demonstrates its capability as a viable alternative to current DPP4 resistance strategies while offering additional structural variation that may influence downstream signaling.

Advances in Mass Spectrometry Fragmentation Techniques for Peptide and Protein Characterization: CID to ETD

Mass spectrometry (MS) has transformed the field of proteomics by enabling the precise analysis of proteins and peptides at the molecular level. The heart of this analysis is the process of fragmentation, which plays a pivotal role in elucidating amino acid sequence, identifying post-translational modifications (PTMs), and characterizing structural features. Peptides and proteins are first ionized and then subjected to fragmentation techniques such as Collision-Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), and Electron-Capture Dissociation (ECD). These techniques induce fragmentation along the peptide backbone, generating characteristic fragment ions (b and y ions, c and z ions) that reflect the sequence of amino acids. The mass-to-charge (m/z) ratios of these fragment ions provide crucial data for de novo sequencing and PTM identification. Furthermore, fragmentation patterns can reveal insights into protein folding, interactions, and dynamics. This article provides the detailed view of mass fragmentation techniques in peptides and proteins, highlighting its importance in advancing proteomic research and its applications in fields ranging from biomarker discovery to drug development.

Poly-Dha Sequences as Pro-polypeptides: An Original Mechanistic Postulate Leads to the Discovery of a Long-Acting Vasodilator KU04212.

The construction of polypeptides was revolutionized by Merrifield's solid-phase synthesis more than half a century ago. Herein, we explore a completely different approach to making peptides. We test an original mechanistic postulate wherein a single peptide made entirely of dehydroalanine (Dha) residues can give rise to regio- and stereodefined peptides by iterative conjugate addition of one- or two-electron nucleophiles. Each nucleophile appends a unique amino acid side chain to the peptide backbone. We show that side chain addition is not random. Side chains are added in one of two ways, in an electrophilicity-gated fashion (most cases) or in a substrate-directed manner, depending on the first nucleophile used in the synthesis. One peptide made in this series, KU04212, a first-in-class polyazole peptide, was found to reduce vascular length density (-17%; p < 0.05) and increase vessel diameter (124%; p < 0.001) in healthy day 6 chick embryos at 24 h post-single dose. It also rescued 75% of the embryos administered a 32-fold lethal dose of ischemia-inducing CoCl2 after 12 h and 12.5% of the embryos after 24 h. In comparison to three mechanistically distinct vasodilators, e.g., isosorbide mononitrate, amlodipine besylate, and prazosin, only KU04212 showed long-acting effects in vivo, making it an enticing lead for the treatment of ischemic disorders.

Innovative On-Resin and in Solution Peptidomimetics Synthesis via Metal-Free Photocatalytic Approach.

Nowadays, peptidomimetics are widely studied, being useful tools in drug discovery and medicinal chemistry. The coupling between a carboxylic acid with an amine to form a peptide bond is the most common reaction to obtain peptides/peptidomimetics. In this work, we have investigated an innovative metal-free photoredox-catalyzed carbamoylation to form peptidomimetics thanks to the reaction between dihydropyridines functionalized with amino acids (or peptide sequences) and differently functionalized imines. As the organic photocatalyst, we used 4CzIPN, a donor-acceptor cyanoarene vastly used in photoredox catalysis. By easily modulating the amino acid (or peptide sequence), which is directly attached to the dihydropyridine, we proposed this key-reaction as new valuable method to obtain peptidomimetics, in situ building the not-natural portion of the sequence. Moreover, we successfully employed this methodology in solid phase peptide synthesis, both inserting the new photoredox-generated amino acid at the end or in the middle of the sequence. Peptides with different lengths and secondary structures were prepared, proving the success of this approach, even in sterically hindered environment. Herein, to the best of our knowledge, we describe the first photocatalytic protocol which allows the building of the peptide backbone, with the possibility of simultaneously inserting a non-coded amino acid in the sequence.

Peptide retention time prediction for electrostatic repulsion-hydrophilic interaction chromatography

Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) is one of the legacy separation tools developed by Dr. Andrew Alpert and has been used for developing unique separation methods of hydrophilic compounds, including peptides. In the past it has been studied using designed peptide libraries to elucidate major features of its separation mechanism, while comprehensive peptide retention modeling for ERLIC is still lacking. In this work we employed a proteomics-derived ∼170,000 peptide retention datasets to evaluate major ERLIC retention features using the framework of our Sequence-Specific Retention Calculator model. The separation conditions were adjusted to obtain a wider proteome coverage, particularly for non-modified peptides, resulting in a superior separation orthogonality for a 2D LC combination with reversed-phase C18 LC-MS in the second dimension. The SSRCalc ERLIC model presents a consistent theme with the existing ERLIC retention mechanism, reflecting a dependence on peptide orientation and the position of charged and hydrophilic residues across the peptide backbone. R2 values of 0.935 and 0.955 accuracy were demonstrated for the standard interpretable SSRCalc model and machine learning algorithm, respectively. The effects of various PTMs on peptide retention were evaluated in this study, covering spontaneous (oxidation, deamidation) and enzymatic (N-terminal acetylation, phosphorylation, glycosylation) modifications.

Theoretical investigation for the proline effect on peptide fragmentation induced by matrix-assisted laser desorption/ionization in-source decay with an oxidizing matrix

The use of an oxidizing matrix for matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) specifically induces the cleavage of Cα-C bonds on the peptide backbone, except for bonds located on the N-terminal side of proline residues. To examine the effect of Pro residues on bond cleavage induced by MALDI-ISD with an oxidizing matrix, the small dipeptides AcAA-NH2 and AcAP-NH2 were used as models. As the fragmentation is initiated by electron transfer from the peptide to the oxidizing matrix, the dissociation chemistry of the cation radical forms of the model peptides [AcAA-NH2]+• and [AcAP-NH2]+• was investigated using quantum chemistry calculations. The [AcAA-NH2]+• can produce fragment ions not only due to Cα–C bond cleavage, but also peptide bond cleavage. Quantum chemistry calculations indicated that peptide bond cleavage of [AcAA-NH2]+• occurs more slowly compared to Cα–C bond cleavage. Instead of Cα–C bond cleavage, the bond on the N-terminal side of Pro residue undergoes peptide bond cleavage during MALDI-ISD with an oxidizing matrix, due to the lack of an amide hydrogen in the Pro residue. The [AcAP-NH2]+• undergoes proton migration from the δ-carbon of the Pro residue. Depending on the proton binding site in the peptide cation radical, the peptide bond cleavage of [AcAP-NH2]+• results in the formation of either [b1]+, and [y1]•, or [a1]• and [y1]+. These theoretical results are consistent with experimental findings, and the newly proposed mechanism involving peptide cation radical formation followed by proton migration provides a more accurate model for the MALDI-ISD processes.

Comparative proteomic analysis of metronidazole-sensitive and resistant Trichomonas vaginalis suggests a novel mode of metronidazole action and resistance

The microaerophilic parasite Trichomonas vaginalis occurs worldwide and causes inflammation of the urogenital tract, especially in women. With 156 million infections annually, trichomoniasis is the most prevalent non-viral sexually transmitted disease. Trichomoniasis is treated with 5-nitroimidazoles, especially metronidazole, which are prodrugs that have to be reduced at their nitro group to be activated. Resistance rates to metronidazole have remained comparably low, but they can be higher in certain areas leading to an increase of refractory cases. Metronidazole resistance in T. vaginalis can develop in vivo in clinical isolates, or it can be induced in the laboratory. Both types of resistance share certain characteristics but differ with regard to the dependence of ambient oxygen to become manifest. Although several candidate factors for metronidazole resistance have been described in the past, e.g. pyruvate:ferredoxin oxidoreductase and ferredoxin or thioredoxin reductase, open questions regarding their role in resistance have remained.In order to address these questions, we performed a proteomic study with metronidazole-sensitive and –resistant laboratory strains, as well as with clinical strains, in order to identify factors causative for resistance. The list of proteins consistently associated with resistance was surprisingly short. Resistant laboratory and clinical strains only shared the downregulation of flavin reductase 1 (FR1), an enzyme previously identified to be involved in resistance. Originally, FR1 was believed to be an oxygen scavenging enzyme, but here we identified it as a ferric iron reductase which produces ferrous iron. Based on this finding and on further experimental evidence as presented herein, we propose a novel mechanism of metronidazole activation which is based on ferrous iron binding to proteins, thereby rendering them susceptible to complex formation with metronidazole. Upon resolution of iron-protein-metronidazole complexes, metronidazole radicals are formed which quickly react with thiols or proteins in the direct vicinity, leading to breaks in the peptide backbone.

Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.