Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

Hemorphins-From Discovery to Functions and Pharmacology.

During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added.

Composites Based on Gellan Gum, Alginate and Nisin-Enriched Lipid Nanoparticles for the Treatment of Infected Wounds.

Due to growing antimicrobial resistance to antibiotics, novel methods of treatment of infected wounds are being searched for. The aim of this research was to develop a composite wound dressing based on natural polysaccharides, i.e., gellan gum (GG) and a mixture of GG and alginate (GG/Alg), containing lipid nanoparticles loaded with antibacterial peptide-nisin (NSN). NSN-loaded stearic acid-based nanoparticles (NP_NSN) were spherical with an average particle size of around 300 nm and were cytocompatible with L929 fibroblasts for up to 500 µg/mL. GG and GG/Alg sponges containing either free NSN (GG + NSN and GG/Alg + NSN) or NP_NSN (GG + NP_NSN and GG/Alg + NP_NSN) were highly porous with a high swelling capacity (swelling ratio above 2000%). Encapsulation of NSN within lipid nanoparticles significantly slowed down NSN release from GG-based samples for up to 24 h (as compared to GG + NSN). The most effective antimicrobial activity against Gram-positive Streptococcus pyogenes was observed for GG + NP_NSN, while in GG/Alg it was decreased by interactions between NSN and Alg, leading to NSN retention within the hydrogel matrix. All materials, except GG/Alg + NP_NSN, were cytocompatible with L929 fibroblasts and did not cause an observable delay in wound healing. We believe that the developed materials are promising for wound healing application and the treatment of bacterial infections in wounds.

Inhibitors of neuropeptide peptidases engaged in pain and drug dependence.

Owing to a broad spectrum of functions performed by neuropeptides, this class of signaling molecules attracts an increasing interest. One of the key steps in the regulation of biological activity of neuropeptides is proteolytic conversion or degradation by proteinases that change or terminate biological activity of native peptides. These enzymes, in turn, are regulated by inhibitors, which play integral role in controlling many metabolic pathways. Thus, the search for selective inhibitors and detailed knowledge on the mechanisms of binding of these substances to enzymes, could be of importance for designing new pharmacological approaches. The aim of this review is to summarize the current knowledge on the inhibitors of enzymes that convert selected groups of neuropeptides, such as dynorphins, enkephalins, substance P and NPFF fragments. The importance of these substances in pathophysiological processes involved in pain and drug addiction, have been discussed. This article is part of the special issue on Neuropeptides.

Mouse single oocyte imaging by MALDI-TOF MS for lipidomics.

Reproductive cells are a very special kind of material for the analysis. Depending on the species, their dimensions allow for the application of mass spectrometry imaging-based techniques to receive a reasonable data for interpretation of their condition without any additional sample preparation steps, except for typical sample preparation characteristic for IMS protocols. A comparison between lipid profiles of oocytes could answer the question of the overall quality of the cells in the function of time or conditions of storage. Even tiny differences in the lipid profiles, but still detectable by bioinformatic analysis, could be crucial for the estimation of the conditions of the cells in various stages of development or aging. In our study, MALDI-TOF/TOF MSI was used to analyze and visualize the single oocytes. We deposited the cells on the transparent indium-tin-oxide (ITO) glass and marked their positions, which allowed for the fast localization of the cells and precise laser targeting in the ion source. We also optimized the usage of different MALDI matrices and different approaches. The proposed way of measurement allows analyzing quite a significant quantity of oocytes in a reasonably short time. During the analysis, the lipid composition of the single cell was successfully estimated in a conventional usage of the MALDI ion source, and the localization of lipids was confirmed by imaging mass spectrometry (IMS) analysis. The observed quantity of the lipids allowed for the application of the LIFT™ technique to obtain MS/MS spectra sufficient for lipids' unambiguous identification. We hope that our idea of the oocyte analysis will help to elucidate chemical changes that accompany different processes in which oocytes are involved. There could be such fascinating phenomena as the oocyte maturation, changes in the lipid components during their storage, and much more.

Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group.

This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease-staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme.

Changes in Protein Glycosylation as a Result of Aptamer Interactions with Cancer Cells.

PURPOSE: Based on the recent aptamer-related breast cancer studies, which indicate the therapeutic role of specific oligonucleotide sequences, experiments have been designed in an attempt to unravel the molecular targets of this mechanism. This article describes the study on glycoproteome changes in breast cancer cells as a result of their interactions with aptamers. EXPERIMENTAL DESIGN: Aberrations in protein glycosylation play an important role in tumorigenesis and influence cancer progression, metastasis, immunoresponse, and chemoresistance, therefore this study is focused on the identification of the alterations in glycan expression on the surface of proteins as a potential and innovative tool for biomedical applications of aptamers in cancer treatment. RESULTS: Two proteins, kinesin-like protein (KI13B) and proliferating cell nuclear antigen (PCNA), have been identified that carry N-glycan epitopes after conjugation with aptamer sequences. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple features of aptamers as an alternative to protein antibodies are utilized for various biomedical applications ranging from biomarker discovery, bioimaging, targeted therapy, drug delivery, and drug pharmacokinetics and biodistribution. Frequently, aptamers bind to their target molecules and modulate their function. Such therapeutic aptamers can modify the biological pathways for treatment of many types of diseases, such as cancer.