Structure and function of methionine aminopeptidases (2023)

In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the methionine primer in at least 70% of newly synthesized polypeptides. MetAPs are commonly classified into Type 1 and Type 2. While prokaryotes and archaea contain only type 1 and type 2 MetAPs, respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published to date only cleave the amino-terminal methionine of substrate peptides. Previous experiments with crude type 2a MetAP isolated fromPyrococci have gone insane(PoohThe MetAP2a cosmid protein library has been shown to cleave leucine in addition to methionine. The authors of this study rejected thePoohMetAP2a activity against leucine substrates and presumably a background reaction contributed by other contaminating proteases. In the current article, using the pure recombinant enzyme, we report that the activity against leucine is actually performed directly by thePoohMetaAP2a. Furthermore, the natural product ovalicin, which is a specific covalent inhibitor of type 2 MetAPs, does not show potent inhibition against thePoohMetaAP2a. Bioinformatic analyzes suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 inPoohMetAP2a) in archaeal MetAP2s positioned in the analogous position. The N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance to the active site and changes orientation to accommodate ovalicin. This slight difference in the orientation of H62 reduces the affinity of ovalicin by 300,000-fold compared to H62hsMetAP2b inhibition. This difference in activity is partially reduced in the N53G mutationPoohMetAP2a.

A method for synthesizing terminal olefins by α-methylenation of aromatic ketones is reported. The reaction was carried out in air using acetonitrile as the carbon source and Selectfluor as a mild oxidant. The scope and versatility of the method were demonstrated using 29 examples. Based on experimental results, an oxidative reaction mechanism promoted by selectfluor is proposed.

The conjugation of functional molecules to peptides is necessary for protein analysis and applications. Sortase A transpeptidase catalyzes the binding reaction between the LPXTG amino acid sequence and polyglycine and enables specific molecular modifications of the peptide sequence. In this study, the preparation of green fluorescent protein fused to pentaglycine (G5-GFP)vonMethionine shortening mediated byEscherichia coliEndogenous methionylaminopeptidase was examined. Several GFP expression vectors with MetGly5 at the N-terminus were constructed and N-terminal sequence analysis of the expressed protein was performedE coliWe're done. When the first codon of the GFP coding sequence was AUG, a mixture of GFP lacking pentaglycine and G5-GFP was obtained. In contrast, when the first AUG codon was replaced with a codon encoding alanine, G5-GFP was obtained uniformly. These results indicated that the position of AUG in the expression vector had a significant impact on the production of polyglycine fused proteins. The results obtained are useful for the production of casting substrates with polyglycineE coli.

Fusarium oxysporumF. sp.cucumbers(FOC) is one of the main fungi infecting cucumber plants, causing significant productivity losses. Phytopathogenic fungi vary in disease severity and degree of aggressiveness, with different modes of infection being driven by specialized proteins involved in signaling pathways leading to pathogenicity, an area that is less understood. In order to understand the spectrum of pathogenicity and virulence of FOC isolates, a comprehensive proteomics study was performed to determine the polypeptide patterns of mycelial, conidial and secreted proteins from highly pathogenic (hp-FOC) and low pathogenic (wp-FOC) to compare isolates. Two-dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS analysis led to the identification of common and unique proteins involved in metabolism and pathogenicity in both fungal isolates. This study can describe the biological and molecular basis of different fungal structures to understand the mechanism of their action in host tissues. Determining proteins that express the pathogen's ability to induce pathogenesis can aid in the detection of specialized inhibitors that suppress the activities of pathogen proteins to fight the disease, which can help reduce overuse of fungicides, the still further increasing. plus productivity, quality and environmental safety.

Aminopeptidases, which are widespread in nature, are one of two major subclasses of exopeptidases, proteolytic enzymes that remove amino acids from the ends of peptides and proteins (the others are carboxypeptidases). As the name suggests, aminopeptidases only attack their substrates at the amino-terminal end. Most remove one amino acid at a time, but a small group cleave two or three residues at a time; these are known as dipeptidyl and tripeptidyl aminopeptidases, respectively. Some enzymes such as acylaminoacylpeptidase and pyroglutamylpeptidase remove derived amino acids, but in general aminopeptidases require an unmodified or free amino group.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has led to an ongoing pandemic. There are currently no clinically approved drugs for COVID-19. Therefore, there is an urgent need to accelerate the development of effective antivirals. Here we discover clioquinol (5-chloro-7-iodo-8-quinolinol (CLQ)), a drug approved by the Food and Drug Administration (FDA) and two of its analogues (7-bromo-5-chloro-8-hydroxyquinoline ( CLBQ14) and 5,7-dichloro-8-hydroxyquinoline (CLCQ)) as potent inhibitors of the cytopathic effect induced by SARS-CoV-2 infectionin-vitro.Besides that,All three compounds showed potent anti-exopeptidase activity against recombinant human angiotensin converting enzyme 2 (rhACE2) and inhibited rhACE2 binding to the SARS-CoV-2 spike protein (RBD). CLQ showed the greatest potency in the low micromolar range, with its antiviral activity showing a strong correlation with inhibition of rhACE2 and rhACE2-RBD interaction. Taken together, our results provide a novel mode of action and molecular target for CLQ and validate this pharmacophore as a promising lead series for the clinical development of potential therapeutics for COVID-19.

Current Biology, Band 27, Heft 20, 2017, S. 3183-3190.e4

Abiotic stress negatively affects plant growth and significantly affects crop yield and quality. While much is known about plant responses, very little is known about initial perceptions of environmental stress at the molecular level. In plants, hypoxia (low oxygen levels that occurs during flooding) is directly mediated by the Cys-Arg/N-terminal pathway of ubiquitin-mediated proteolysis through the oxygen-dependent degradation of group VII ethylene response factor transcription factors (ERFVIIs) via the amino-terminal ( Nt-)cysteine ​​[1,2]. UseArabidopsis(Arabidopsis thaliana) and barley (ordinary barley) we show that the signaling pathway regulates plant responses to multiple abiotic stresses. InArabidopsis, genetic analyzes showed that the response to these stresses is controlled by regulation of the N-end rule ofERFVIIFunction. Oxygen detection via the Cys-Arg/N-End rule in higher eukaryotes is linked to nitric oxide (NO) detection by a unique mechanism [3,4]. In plants, the main mechanism of NO synthesis is via NITRATE REDUCTASE (NR), a nitrogen assimilation enzyme.5]. Here we identified a negative relationship between NR activity and NO levels and the stabilization of an artificial Nt-Cys substrate andERFVIIFunction in response to environmental changes. Also, we show thatERFVIIs amplify abiotic stress responses through physical and genetic interactions with the ATPase BRAHMA, which remodels chromatin. We propose that plants recognize multiple abiotic stresses via the Cys-Arg/N-End pathway, either directly (via oxygen detection) or indirectly (via NO detection after NR activity). This unique mechanism can therefore integrate environment and response to enhance plant survival.

Biochemie, Band 114, 2015, S. 134-146

N-terminal protein modifications correspond to the first modifications that, in principle, any protein can undergo before translation by the ribosome is complete. This class of essential modifications can differ in nature or function and are catalyzed by a variety of dedicated enzymes. Here we give an overview of the current status of the most important N-terminal cotranslational modifications, with a special focus on their catalysts, which belong to the metalloprotease and acyltransferase clans. The first of these modifications corresponds to the excision of the N-terminal methionine, a ubiquitous and essential process leading to the removal of the first methionine. N-alpha acetylation also occurs in all kingdoms, although its extent appears to be significantly increased in higher eukaryotes. Finally, N-myristoylation is a crucial pathway that only exists in eukaryotes. Recent studies on how some of these co-translational modifiers might function in close proximity to the ribosome provide new information on exactly when these modifications occur on the elongating nascent chain and the interaction with other ribosome biogenesis factors responsible for nascent chains are. A comprehensive overview of recent advances in the field of N-terminal protein modifications can be found here.

Journal of Biological Chemistry, Band 294, Ausgabe 12, 2019, S. 4464-4476

All organisms begin protein synthesis with methionine (Met). The Met primer resulting from the resulting proteins is irreversibly processed by Met aminopeptidases (MetAPs). Excision of the N-terminal (Nt) Met (NME) is an evolutionarily conserved and essential process that acts on up to two-thirds of proteins. However, the universal function of NME is still largely unknown. MetAPs have a known processing preference for Nt-Met with Ala, Ser, Gly, Thr, Cys, Pro, or Val at position 2, but use CHX chase assays to assess protein degradation in yeast cells, as well as protein binding assays and RT-qPCR, we show here, that NME also occurs in nascent proteins that contain Met-Asn or Met-Gln at their N-terminus. We found that NME destabilizes at these tertiary Nt residues (Asn or Gln) of the Arg/N-terminal regulatory pathway that proteins ​according to the composition of their Nt residues. We also identified a yeast DNA repair protein, MQ-Rad16, bearing a Met-Gln N-terminus, and a human tropomyosin receptor kinase (TFG) gene protein, MN-TFG, bearing a Met-Asn N-terminus as physiological Arg carries /N final rule substrates that are processed by MetAP. Furthermore, we show that the loss of components of the Arg/N-end pathway substantially suppresses the growth defects ofnow 20Δ Yeast cells lacking the catalytic subunit of NatB-Nt acetylase at 37 °C. In summary, the results of our study demonstrate that NME is an important upstream step for the creation of Arg/N-final rule substrates containing tertiary destabilizing residues.live.

Journal of Molecular Biology, Band 431, Ausgabe 7, 2019, S. 1426-1439

Trends in Biochemical Sciences, Band 46, Ausgabe 1, 2021, S. 15-27

N-terminal acetylation (NTA) is one of the most common protein modifications, occurring in most eukaryotic proteins but much less common in bacterial and archaeal proteins. This modification is performed by a family of enzymes called N-terminal acetyltransferases (NATs). To date, 12 NATs have been identified, harboring different compositions, substrate specificities, and in some cases modes of regulation. Recent structural and biochemical analyzes of NAT proteins allow comparison of their molecular mechanisms and modes of regulation, which are described here. Although they share an evolutionarily conserved fold and related catalytic mechanism, each catalytic subunit uses unique elements to mediate substrate-specific activity and uses NAT-like specific regulatory and helper subunits for their cellular functions.

Bioorganic & Medicinal Chemistry Letters, Band 27, Ausgabe 3, 2017, S. 551-556

The natural substance fumagillin1and derivatives such as TNP-4702or beloranib3bind irreversibly to methionine aminopeptidase 2 (MetAP-2). This enzyme is crucial for protein maturation and plays a key role in angiogenesis. In this article, we describe the synthesis, binding affinity of MetAP-2, and structural analysis of reversible MetAP-2 inhibitors. The optimization of the enzymatic activity of the screen reached 10 (CI₅₀: 1μM) led to the strongest compound27(CI₅₀: 0.038 μM), with a simultaneous improvement in the LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the imidazole ring of the His339 side chain, which is coplanar between the imidazole of His331 and the aryl ether moiety attached to the purine backbone. Systematic modification and reduction of the H-bonding capacity of this metal-binding moiety induced an unexpected 180° rotation of the triazolo[1,5-A] bicyclic mold of pyrimidine.