Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture

Nusinersen mitigates neuroinflammation in severe spinal muscular atrophy patients

Background: Neuroinflammation contributes to the onset and progression of neurodegenerative diseases, but has not been specifically investigated in patients affected by severe and milder forms of spinal muscular atrophy (SMA). Methods: In this two-center retrospective study, we investigated signatures of neuroinflammation in forty-eight pediatric male and female SMA1 (n = 18), male and female SMA2 (n = 19), and female SMA3 (n = 11) patients, as well as in a limited number of male and female non-neurological control subjects (n = 4). We employed a Bio-Plex multiplex system based on xMAP technology and performed targeted quantitative analysis of a wide range of pro- and anti-inflammatory cytokines (chemokines, interferons, interleukins, lymphokines and tumor necrosis factors) and neurotrophic factors in the cerebrospinal fluid (CSF) of the study cohort before and after Nusinersen treatment at loading and maintenance stages. Results: We find a significant increase in the levels of several pro-inflammatory cytokines (IL-6, IFN-γ, TNF-α, IL-2, IL-8, IL-12, IL-17, MIP-1α, MCP-1, and Eotaxin) and neurotrophic factors (PDGF-BB and VEGF) in the CSF of SMA1 patients relative to SMA2 and SMA3 individuals, who display levels in the range of controls. We also find that treatment with Nusinersen significantly reduces the CSF levels of some but not all of these neuroinflammatory molecules in SMA1 patients. Conversely, Nusinersen increases the CSF levels of proinflammatory G-CSF, IL-8, MCP-1, MIP-1α, and MIP-1β in SMA2 patients and decreases those of anti-inflammatory IL-1ra in SMA3 patients. Conclusions: These findings highlight signatures of neuroinflammation that are specifically associated with severe SMA and the neuro-immunomodulatory effects of Nusinersen therapy

Myoglobin from Atlantic and Tinker mackerels: Purification, characterization and its possible use as a molecular marker

Here, we report the characterization (purification, autoxidation rate, pseudoperoxidase activity) and amino acid sequence determination of S. scombrus (Atlantic mackerel) and S. colias (Tinker mackerel) mioglobins (Mbs), considering the increasing consumption of fresh and canned mackerel meat and Mb implication in meat storage (e.g.: browning and lipid oxidation). We found that Atlantic mackerel Mb has major autoxidation rate (0.204 +/- 0.013 h-1) compared to Tinker mackerel Mb (0.140 +/- 0.009 h-1), while the pseudoperoxidase activity is major for Tinker mackerel (Km: 87.71 +/- 7.19 mu M; kcat: 0.32 s-1) Mb with respect to Atlantic mackerel (Km: 96.08 +/- 6.91 mu M; kcat: 0.50 s-1). These functional differences are confirmed by primary structure determination, in which six amino acid substitutions are found, with the first N-terminal amino acid residue acetylated. Furthermore, we predicted by AphaFold 3D model both fish Mbs and used them to investigate the possible structural differences. In addition, phylogenetic analysis using Mb sequences from Scombridae family confirms that Atlantic and Tinker mackerels are two distinct species. Finally, an analytic qualitative RP-HPLC method to distinguish S. scombrus and S. colias specimens was developed considering the different retention times of the two mackerel apoMbs

Molecular Fingerprint of Human Pathological Synoviocytes in Response to Extractive Sulfated and Biofermentative Unsulfated Chondroitins

Pharma-grade extractive chondroitin sulfate (CS) is widely used for osteoarthritis (OA) treatment. Recently, unsulfated biofermentative chondroitin (BC) proved positive effects in OA in vitro model. This study, based on primary pathological human synoviocytes, aimed to analyze, by a multiplex assay, a panel of OA-related biomarkers in response to short-term treatments with bovine (CSb), pig (CSp) and fish (CSf) chondroitins, in comparison to BC. As expected, all samples had anti-inflammatory properties, however CSb, CSf and especially BC affected more cytokines and chemokines. Based on these results and molecular weight similarity, CSf and BC were selected to further explore the synoviocytes’ response. In fact, Western blot analyses showed CSf and BC were comparable, downregulating OA-related biomarkers such as the proteins mTOR, NF-kB, PTX-3 and COMP-2. Proteomic analyses, performed by applying a nano-LC-MS/MS TMT isobaric labelling-based approach, displayed the modulation of both common and distinct molecules to chondroitin treatments. Thus, CSf and BC modulated the biological mediators involved in the inflammation cascade, matrix degradation/remodeling, glycosaminoglycans’ synthesis and cellular homeostasis. This study helps in shedding light on different molecular mechanisms related to OA disease that may be potentially affected not only by animal-source chondroitin sulfate but also by unsulfated biofermentative chondroitin

Deciphering Molecular Determinants Underlying Penicillium digitatum’s Response to Biological and Chemical Antifungal Agents by Tandem Mass Tag (TMT)-Based High-Resolution LC-MS/MS

Penicillium digitatum is a widespread pathogen responsible for the postharvest decay of citrus, one of the most economically important crops worldwide. Currently, chemical fungicides are still the main strategy to control the green mould disease caused by the fungus. However, the increasing selection and proliferation of fungicide-resistant strains require more efforts to explore new alternatives acting via new or unexplored mechanisms for postharvest disease management. To date, several non-chemical compounds have been investigated for the control of fungal pathogens. In this scenario, understanding the molecular determinants underlying P. digitatum’s response to biological and chemical antifungals may help in the development of safer and more effective non-chemical control methods. In this work, a proteomic approach based on isobaric labelling and a nanoLC tandem mass spectrometry approach was used to investigate molecular changes associated with P. digitatum’s response to treatments with α-sarcin and beetin 27 (BE27), two proteins endowed with antifungal activity. The outcomes of treatments with these biological agents were then compared with those triggered by the commonly used chemical fungicide thiabendazole (TBZ). Our results showed that differentially expressed proteins mainly include cell wall-degrading enzymes, proteins involved in stress response, antioxidant and detoxification mechanisms and metabolic processes such as thiamine biosynthesis. Interestingly, specific modulations in response to protein toxins treatments were observed for a subset of proteins. Deciphering the inhibitory mechanisms of biofungicides and chemical compounds, together with understanding their effects on the fungal physiology, will provide a new direction for improving the efficacy of novel antifungal formulations and developing new control strategies

Differential Secretome Profiling of Human Osteoarthritic Synoviocytes Treated with Biotechnological Unsulfated and Marine Sulfated Chondroitins

Symptomatic slow-acting drugs (SYSADOA) are increasingly used as effective therapies for osteoarthritis, representing an attractive alternative to analgesics or non-steroidal anti-inflammatory drugs to relieve disease symptoms. Pharmaceutical preparations of chondroitin sulfate, derived from animal sources, alone or in combination with glucosamine sulfate, are widely recognized for their beneficial effect on osteoarthritis treatment. A growing interest has also been devoted to understanding the molecular mechanisms modulated by SYSADOA using -omic strategies, most of which rely on chondrocytes as a model system. In this work, by using an integrated strategy based on unbiased proteomics and targeted cytokine profiling by a multiplexed protein array, we identified differences in the secretomes of human osteoarthritic synoviocytes in response to biotechnological unsulfated, and marine sulfated chondroitins treatments. The combined strategy allowed the identification of candidate proteins showing both common and distinct regulation responses to the two treatments of chondroitins. These molecules, mainly belonging to ECM proteins, enzymes, enzymatic inhibitors and cytokines, are potentially correlated to treatment outcomes. Overall, the present results provide an integrated overview of protein changes in human osteoarthritic synoviocytes secretome associated to different chondroitin treatments, thus improving current knowledge of the biochemical effects driven by these drugs potentially involved in pathways associated to osteoarthritis pathogenesis

The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex

Background Imprinting Control Regions (ICRs) are CpG-rich sequences acquiring differential methylation in the female and male germline and maintaining it in a parental origin-specific manner in somatic cells. Despite their expected high mutation rate due to spontaneous deamination of methylated cytosines, ICRs show conservation of CpG-richness and CpG-containing transcription factor binding sites in mammalian species. However, little is known about the mechanisms contributing to the maintenance of a high density of methyl CpGs at these loci. Results To gain functional insights into the mechanisms for maintaining CpG methylation, we sought to identify the proteins binding the methylated allele of the ICRs by determining the interactors of ZFP57 that recognizes a methylated hexanucleotide motif of these DNA regions in mouse ESCs. By using a tagged approach coupled to LC-MS/MS analysis, we identified several proteins, including factors involved in mRNA processing/splicing, chromosome organization, transcription and DNA repair processes. The presence of the post-replicative mismatch-repair (MMR) complex components MSH2 and MSH6 among the identified ZFP57 interactors prompted us to investigate their DNA binding profile by chromatin immunoprecipitation and sequencing. We demonstrated that MSH2 was enriched at gene promoters overlapping unmethylated CpG islands and at repeats. We also found that both MSH2 and MSH6 interacted with the methylated allele of the ICRs, where their binding to DNA was mediated by the ZFP57/KAP1 complex. Conclusions Our findings show that the MMR complex is concentrated on gene promoters and repeats in mouse ESCs, suggesting that maintaining the integrity of these regions is a primary function of highly proliferating cells. Furthermore, the demonstration that MSH2/MSH6 are recruited to the methylated allele of the ICRs through interaction with ZFP57/KAP1 suggests a role of the MMR complex in the maintenance of the integrity of these regulatory regions and evolution of genomic imprinting in mammalian species

ZBTB2 protein is a new partner of the Nucleosome Remodeling andDeacetylase (NuRD) complex

ZBTB2 is a protein belonging to the BTB/POZ zinc-finger family whose members typically contain a BTB/POZ do-main at the N-terminus and several zinc-finger domains at the C-terminus. Studies have been carried out to dis-close the role of ZBTB2 in cell proliferation, in human cancers and in regulating DNA methylation. Moreover,ZBTB2 has been also described as an ARF, p53 and p21 gene repressor as well as an activator of genes modulatingpluripotency. In this scenario, ZBTB2 seems to play many functions likely associated with other proteins. Here wereport a picture of the ZBTB2 protein partners in U87MG cell line, identified by high-resolution mass spectrom-etry (MS) that highlights the interplay between ZBTB2 and chromatin remodeling multiprotein complexes.In particular, our analysis reveals the presence, as ZBTB2 candidate interactors, of SMARCA5 and BAZ1B compo-nents of the chromatin remodeling complex WICH and PBRM1, a subunit of the SWI/SNF complex. Intriguingly,we identified all the subunits of the NuRD complex among the ZBTB2 interactors. By co-immunoprecipitation ex-periments and ChIP-seq analysis we definitely identify ZBTB2 as a new partner of the NuRD complex

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes