@article{MTMT:34781847, title = {Unveiling the Oxazolidine Character of Pseudoproline Derivatives by Automated Flow Peptide Chemistry}, url = {https://m2.mtmt.hu/api/publication/34781847}, author = {Szaniszló, Szebasztián and Csámpai, Antal and Horváth, Dániel and Tomecz, Richárd and Farkas, Viktor and Perczel, András}, doi = {10.3390/ijms25084150}, journal-iso = {INT J MOL SCI}, journal = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, volume = {25}, unique-id = {34781847}, issn = {1661-6596}, abstract = {Pseudoproline derivatives such as Thr(ΨPro)-OH are commonly used in peptide synthesis to reduce the likelihood of peptide aggregation and to prevent aspartimide (Asi) formation during the synthesis process. In this study, we investigate notable by-products such as aspartimide formation and an imine derivative of the Thr(ΨPro) moiety observed in flow peptide chemistry synthesis. To gain insight into the formation of these unexpected by-products, we design a series of experiments. Furthermore, we demonstrate the oxazolidine character of the pseudoproline moiety and provide plausible mechanisms for the two-way ring opening of oxazolidine leading to these by-products. In addition, we present evidence that Asi formation appears to be catalyzed by the presence of the pseudoproline moiety. These observed side reactions are attributed to elevated temperature and pressure; therefore, caution is advised when using ΨPro derivatives under such harsh conditions. In addition, we propose a solution whereby thermodynamically controlled Asi formation can be kinetically prevented.}, year = {2024}, eissn = {1422-0067}, orcid-numbers = {Szaniszló, Szebasztián/0000-0002-8929-0635; Csámpai, Antal/0000-0003-2107-7309; Horváth, Dániel/0000-0001-8239-3933; Farkas, Viktor/0000-0002-8815-2783; Perczel, András/0000-0003-1252-6416} } @article{MTMT:34199131, title = {Amide isomerization pathways: Electronic and structural background of protonation- and deprotonation-mediated cis-trans interconversions}, url = {https://m2.mtmt.hu/api/publication/34199131}, author = {Kelemen, Ádám András and Perczel, András and Horváth, Dániel and Jákli, Imre}, doi = {10.1063/5.0165772}, journal-iso = {J CHEM PHYS}, journal = {JOURNAL OF CHEMICAL PHYSICS}, volume = {159}, unique-id = {34199131}, issn = {0021-9606}, abstract = {The cis-trans isomerization of amide bonds leads to wide range of structural and functional changes in proteins and can easily be the rate-limiting step in folding. The trans isomer is thermodynamically more stable than the cis, nevertheless the cis form can play a role in biopolymers’ function. The molecular system of N-methylacetamide · 2H2O is complex enough to reveal energetics of the cis-trans isomerization at coupled cluster single-double and coupled cluster single–double and perturbative triple [CCSD(T)] levels of theory. The cis-trans isomerization cannot be oversimplified by a rotation along ω, since this rotation is coupled with the N-atom pyramidal inversion, requesting the introduction of a second dihedral angle “α.” Full f(ω,α) potential energy surfaces of the different amide protonation states, critical points and isomerization reaction paths were determined, and the barriers of the neutral, O-protonated and N-deprotonated amides were found too high to allow cis-trans interconversion at room temperature: ∼85, ∼140, and ∼110 kJ mol−1, respectively. For the N-protonated amide bond, the cis form (ω = 0°) is a maximum rather than a minimum, and each ω state is accessible for less than ∼10 kJ mol−1. Here we outline a cis-trans isomerization pathway with a previously undescribed low energy transition state, which suggests that the proton is transferred from the more favorable O- to the N-protonation site with the aid of nearby water molecules, allowing the trans → cis transition to occur at an energy cost of ≤11.6 kJ mol−1. Our results help to explain why isomerase enzymes operate via protonated amide bonds and how N-protonation of the peptide bond occurs via O-protonation.}, year = {2023}, eissn = {1089-7690}, orcid-numbers = {Perczel, András/0000-0003-1252-6416; Horváth, Dániel/0000-0001-8239-3933} } @article{MTMT:34119363, title = {Downregulation of transposable elements extends lifespan in Caenorhabditis elegans}, url = {https://m2.mtmt.hu/api/publication/34119363}, author = {Sturm, Ádám and Saskői, Éva and Hotzi, Bernadette and Tarnóci, Anna and Barna, János and Bodnár, Ferenc and Sharma, Himani and Kovács, Tibor and Ari, Eszter and Weinhardt, Nóra and Kerepesi, Csaba and Perczel, András and Ivics, Zoltán and Vellai, Tibor}, doi = {10.1038/s41467-023-40957-9}, journal-iso = {NAT COMMUN}, journal = {NATURE COMMUNICATIONS}, volume = {14}, unique-id = {34119363}, issn = {2041-1723}, abstract = {Mobility of transposable elements (TEs) frequently leads to insertional mutations in functional DNA regions. In the potentially immortal germline, TEs are effectively suppressed by the Piwi-piRNA pathway. However, in the genomes of ageing somatic cells lacking the effects of the pathway, TEs become increasingly mobile during the adult lifespan, and their activity is associated with genomic instability. Whether the progressively increasing mobilization of TEs is a cause or a consequence of ageing remains a fundamental problem in biology. Here we show that in the nematode Caenorhabditis elegans , the downregulation of active TE families extends lifespan. Ectopic activation of Piwi proteins in the soma also promotes longevity. Furthermore, DNA N 6 -adenine methylation at TE stretches gradually rises with age, and this epigenetic modification elevates their transcription as the animal ages. These results indicate that TEs represent a novel genetic determinant of ageing, and that N 6 -adenine methylation plays a pivotal role in ageing control.}, year = {2023}, eissn = {2041-1723}, orcid-numbers = {Sturm, Ádám/0000-0002-9515-8761; Saskői, Éva/0000-0001-6691-8576; Hotzi, Bernadette/0000-0003-1433-6843; Barna, János/0000-0002-9242-0939; Bodnár, Ferenc/0000-0001-9630-1315; Sharma, Himani/0000-0001-6947-9281; Kovács, Tibor/0000-0002-0632-9128; Ari, Eszter/0000-0001-7774-1067; Kerepesi, Csaba/0000-0001-9541-246X; Perczel, András/0000-0003-1252-6416; Ivics, Zoltán/0000-0002-7803-6658; Vellai, Tibor/0000-0002-3520-2572} } @article{MTMT:34084209, title = {Polymorphic amyloid nanostructures of hormone peptides involved in glucose homeostasis display reversible amyloid formation}, url = {https://m2.mtmt.hu/api/publication/34084209}, author = {Horváth, Dániel and Dürvanger, Zsolt and Karancsiné Menyhárd, Dóra and Sulyok-Eiler, Máté and Bencs, Fruzsina and Gyulai, Gergő and Horváth, Péter and Taricska, Nóra and Perczel, András}, doi = {10.1038/s41467-023-40294-x}, journal-iso = {NAT COMMUN}, journal = {NATURE COMMUNICATIONS}, volume = {14}, unique-id = {34084209}, issn = {2041-1723}, abstract = {A large group of hormones are stored as amyloid fibrils in acidic secretion vesicles before they are released into the bloodstream and readopt their functional state. Here, we identify an evolutionarily conserved hexapeptide sequence as the major aggregation-prone region (APR) of gastrointestinal peptides of the glucagon family: xFxxWL. We determine nine polymorphic crystal structures of the APR segments of glucagon-like peptides 1 and 2, and exendin and its derivatives. We follow amyloid formation by CD, FTIR, ThT assays, and AFM. We propose that the pH-dependent changes of the protonation states of glutamate/aspartate residues of APRs initiate switching between the amyloid and the folded, monomeric forms of the hormones. We find that pH sensitivity diminishes in the absence of acidic gatekeepers and amyloid formation progresses over a broad pH range. Our results highlight the dual role of short aggregation core motifs in reversible amyloid formation and receptor binding.}, year = {2023}, eissn = {2041-1723}, orcid-numbers = {Horváth, Dániel/0000-0001-8239-3933; Dürvanger, Zsolt/0000-0002-2652-4916; Karancsiné Menyhárd, Dóra/0000-0002-0095-5531; Sulyok-Eiler, Máté/0000-0002-3968-8776; Bencs, Fruzsina/0009-0003-9246-2228; Gyulai, Gergő/0000-0002-1352-2014; Horváth, Péter/0000-0001-7149-4173; Taricska, Nóra/0000-0002-9721-953X; Perczel, András/0000-0003-1252-6416} } @article{MTMT:34081394, title = {NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg2+ Ion}, url = {https://m2.mtmt.hu/api/publication/34081394}, author = {Gadanecz, Márton and Fazekas, Zsolt and Pálfy, Gyula and Karancsiné Menyhárd, Dóra and Perczel, András}, doi = {10.3390/ijms241512101}, journal-iso = {INT J MOL SCI}, journal = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, volume = {24}, unique-id = {34081394}, issn = {1661-6596}, abstract = {In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg2+-free and Mg2+-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg2+-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg2+-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an “NMR ensemble” based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg2+-bound and Mg2+-free states of G12C KRAS/GDP.}, year = {2023}, eissn = {1422-0067}, orcid-numbers = {Gadanecz, Márton/0009-0009-8076-7597; Fazekas, Zsolt/0000-0001-5007-4807; Pálfy, Gyula/0000-0003-1590-5331; Karancsiné Menyhárd, Dóra/0000-0002-0095-5531; Perczel, András/0000-0003-1252-6416} } @article{MTMT:34078730, title = {Inhibitor Design Strategy for Myostatin: Dynamics and Interaction Networks Define the Affinity and Release Mechanisms of the Inhibited Complexes}, url = {https://m2.mtmt.hu/api/publication/34078730}, author = {Nagy-Fazekas, Dóra and Fazekas, Zsolt and Taricska, Nóra and Stráner, Pál and Karancsiné Menyhárd, Dóra and Perczel, András}, doi = {10.3390/molecules28155655}, journal-iso = {MOLECULES}, journal = {MOLECULES}, volume = {28}, unique-id = {34078730}, issn = {1420-3049}, abstract = {Myostatin, an important negative regulator of muscle mass, is a therapeutic target for muscle atrophic disorders such as muscular dystrophy. Thus, the inhibition of myostatin presents a strategy to treat these disorders. It has long been established that the myostatin prodomain is a strong inhibitor of the mature myostatin, and the minimum peptide of the prodomain—corresponding to the α1-helix of its lasso-region—responsible for the inhibitory efficiency was defined and characterized as well. Here we show that the minimum peptide segment based on the growth differentiation factor 11 (GDF11), which we found to be more helical in its stand-alone solvated stfate than the similar segment of myostatin, is a promising new base scaffold for inhibitor design. The proposed inhibitory peptides in their solvated state and in complex with the mature myostatin were analyzed by in silico molecule modeling supplemented with the electronic circular dichroism spectroscopy measurements. We defined the Gaussian–Mahalanobis mean score to measure the fraction of dihedral angle-pairs close to the desired helical region of the Ramachandran-plot, carried out RING analysis of the peptide-protein interaction networks and characterized the internal motions of the complexes using our rigid-body segmentation protocol. We identified a variant—11m2—that is sufficiently ordered both in solvent and within the inhibitory complex, forms a high number of contacts with the binding-pocket and induces such changes in its internal dynamics that lead to a rigidified, permanently locked conformation that traps this peptide in the binding site. We also showed that the naturally evolved α1-helix has been optimized to simultaneously fulfill two very different roles: to function as a strong binder as well as a good leaving group. It forms an outstanding number of non-covalent interactions with the mature core of myostatin and maintains the most ordered conformation within the complex, while it induces independent movement of the gate-keeper β-hairpin segment assisting the dissociation and also results in the least-ordered solvated form which provides extra stability for the dissociated state and discourages rebinding.}, year = {2023}, eissn = {1420-3049}, orcid-numbers = {Taricska, Nóra/0000-0002-9721-953X; Stráner, Pál/0000-0003-2240-8501; Karancsiné Menyhárd, Dóra/0000-0002-0095-5531; Perczel, András/0000-0003-1252-6416} } @article{MTMT:34064337, title = {The regulation of the nephrin-nephrin distance by podocin mediates the interallelic interactions of the NPHS2 R229Q variant}, url = {https://m2.mtmt.hu/api/publication/34064337}, author = {Antal, Violetta and Schay, Gusztav and Kétszeri, Máté Csaba and Ungvari-Veres, Anita and Paszty, Katalin and Kellermayer, Miklos and Karancsiné Menyhárd, Dóra and Tory, Kálmán}, journal-iso = {PEDIATR NEPHROL}, journal = {PEDIATRIC NEPHROLOGY}, volume = {38}, unique-id = {34064337}, issn = {0931-041X}, year = {2023}, eissn = {1432-198X}, pages = {2262-2263}, orcid-numbers = {Antal, Violetta/0000-0001-8658-6329; Kétszeri, Máté Csaba/0000-0001-7786-9837; Karancsiné Menyhárd, Dóra/0000-0002-0095-5531; Tory, Kálmán/0000-0002-0316-6212} } @article{MTMT:34036752, title = {Acetyl group for proper protection of β-sugar-amino acids used in SPPS}, url = {https://m2.mtmt.hu/api/publication/34036752}, author = {Varga, István and Goldschmidt Gőz, Viktória and Pintér, István and Csámpai, Antal and Perczel, András}, doi = {10.1007/s00726-023-03278-1}, journal-iso = {AMINO ACIDS}, journal = {AMINO ACIDS}, volume = {55}, unique-id = {34036752}, issn = {0939-4451}, abstract = {The synthesis of d- glucosamine-1-carboxylic acid based β-sugar amino acids (β-SAAs) is typically performed in nine consecutive steps via an inefficient OAc → Br → CN conversion protocol with low overall yield. Here, we present the improved and more efficient synthesis of both Fmoc-GlcAPC-OH and Fmoc-GlcAPC(Ac)-OH, β-SAAs consisting of only 4–5 synthetic steps. Their active ester and amide bond formation with glycine methyl ester (H-Gly-OMe) was completed and monitored by 1 H NMR. The stability of the pyranoid OHs protecting the acetyl groups was investigated under three different Fmoc cleavage conditions and was found to be satisfactory even at high piperidine concentration (e.g. 40%). We designed a SPPS protocol using Fmoc-GlcAPC(Ac)-OH to produce model peptides Gly-β-SAA-Gly as well as Gly-β-SAA-β-SAA-Gly with high coupling efficiency. The products were deacetylated using the Zemplén method, which allows the hydrophilicity of a building block and/or chimera to be fine-tuned, even after the polypeptide chain has already been synthesized.}, year = {2023}, eissn = {1438-2199}, pages = {969-979}, orcid-numbers = {Csámpai, Antal/0000-0003-2107-7309; Perczel, András/0000-0003-1252-6416} } @article{MTMT:34014650, title = {Improved Acylation of Pseudoproline: Masked Threonine in Flow Peptide Chemistry}, url = {https://m2.mtmt.hu/api/publication/34014650}, author = {Szaniszló, Szebasztián and Ferentzi, Kristóf and Perczel, András and Farkas, Viktor}, doi = {10.1021/acs.oprd.3c00029}, journal-iso = {ORG PROCESS RES DEV}, journal = {ORGANIC PROCESS RESEARCH & DEVELOPMENT}, volume = {27}, unique-id = {34014650}, issn = {1083-6160}, year = {2023}, eissn = {1520-586X}, pages = {1053-1060}, orcid-numbers = {Szaniszló, Szebasztián/0000-0002-8929-0635; Perczel, András/0000-0003-1252-6416; Farkas, Viktor/0000-0002-8815-2783} } @article{MTMT:33715979, title = {A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System}, url = {https://m2.mtmt.hu/api/publication/33715979}, author = {Nagy-Fazekas, Dóra and Stráner, Pál and Ecsédi, Péter and Taricska, Nóra and Borbély, Adina Noémi and Nyitray, László and Perczel, András}, doi = {10.3390/bioengineering10030389}, journal-iso = {BIOENGINEERING-BASEL}, journal = {BIOENGINEERING}, volume = {10}, unique-id = {33715979}, abstract = {Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an Escherichia coli protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a Kd of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants.}, year = {2023}, eissn = {2306-5354}, orcid-numbers = {Stráner, Pál/0000-0003-2240-8501; Ecsédi, Péter/0000-0002-4700-125X; Taricska, Nóra/0000-0002-9721-953X; Borbély, Adina Noémi/0000-0002-5506-6555; Nyitray, László/0000-0003-4717-5994; Perczel, András/0000-0003-1252-6416} }