Neuroprotective effects on microglia and insights into the structure–activity relationship of an antioxidant peptide isolated from Pelophylax perezi

© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly citedTryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH2 ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia in the framework of the project POCI-01-0145-FEDER-031158 – PTDC/BII-BIO/31158/2017. The authors would like to thank the participation and scientific support of the Unit projects UIDB/50006/2020 | UIDP/50006/2020, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Universal Faixa ‘B’ (grant number 32103/2018-0). A.P. is a recipient of a post-doctoral grant from the project PTDC/BII-BIO/31158/2017. The authors would like to thank the researcher Roberto Resendes (CiBio, University of the Azores, Ponta Delgada, São Miguel, Azores, Portugal) for the logistical support in the collection of samples. C.P.A acknowledges FCT-MCTES fellowship PD/BD/136860/2018. A.B.-N. and F.C.D.A.L. acknowledge CNPq (grants 420449/2018-3 and 428211/2018-6) for financial support.info:eu-repo/semantics/publishedVersio

Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

We analyzed a multi-drug resistant (MR) HIV-1 re- verse transcriptase (RT), subcloned from a patient- derived subtype CRF02 AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azi- dothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to an- tagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most signif- icant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimi- nation, indicating that the two mechanisms are mu- tually exclusive and that the Q151M pathway is ob- viously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, ad- ditionally harboring the TAM K70R and the rever- sions M151Q as well as R65K since K65R antago- nizes excision. MR-R65K-K70R-M151Q was compe- tent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still pro- moted discrimination. To tackle the multi-drug resis- tance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors be- longing to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs

Exploring transcriptional signalling mediated by OsWRKY13, a potential regulator of multiple physiological processes in rice

BACKGROUND Rice transcription regulator OsWRKY13 influences the functioning of more than 500 genes in multiple signalling pathways, with roles in disease resistance, redox homeostasis, abiotic stress responses, and development. RESULTS To determine the putative transcriptional regulation mechanism of OsWRKY13, the putative cis-acting elements of OsWRKY13-influenced genes were analyzed using the whole genome expression profiling of OsWRKY13-activated plants generated with the Affymetrix GeneChip Rice Genome Array. At least 39 transcription factor genes were influenced by OsWRKY13, and 30 of them were downregulated. The promoters of OsWRKY13-upregulated genes were overrepresented with W-boxes for WRKY protein binding, whereas the promoters of OsWRKY13-downregulated genes were enriched with cis-elements putatively for binding of MYB and AP2/EREBP types of transcription factors. Consistent with the distinctive distribution of these cis-elements in up- and downregulated genes, nine WRKY genes were influenced by OsWRKY13 and the promoters of five of them were bound by OsWRKY13 in vitro; all seven differentially expressed AP2/EREBP genes and six of the seven differentially expressed MYB genes were suppressed by in OsWRKY13-activated plants. A subset of OsWRKY13-influenced WRKY genes were involved in host-pathogen interactions. CONCLUSION These results suggest that OsWRKY13-mediated signalling pathways are partitioned by different transcription factors. WRKY proteins may play important roles in the monitoring of OsWRKY13-upregulated genes and genes involved in pathogen-induced defence responses, whereas MYB and AP2/EREBP proteins may contribute most to the control of OsWRKY13-downregulated genes.This work was supported by grants from the National Program of High Technology Development of China, the National Program on the Development of Basic Research in China, and the National Natural Science Foundation of China

The Plant Short-Chain Dehydrogenase (SDR) superfamily:genome-wide inventory and diversification patterns

Background Short-chain dehydrogenases/reductases (SDRs) form one of the largest and oldest NAD(P)(H) dependent oxidoreductase families. Despite a conserved 'Rossmann-fold' structure, members of the SDR superfamily exhibit low sequence similarities, which constituted a bottleneck in terms of identification. Recent classification methods, relying on hidden-Markov models (HMMs), improved identification and enabled the construction of a nomenclature. However, functional annotations of plant SDRs remain scarce. Results Wide-scale analyses were performed on ten plant genomes. The combination of hidden Markov model (HMM) based analyses and similarity searches led to the construction of an exhaustive inventory of plant SDR. With 68 to 315 members found in each analysed genome, the inventory confirmed the over-representation of SDRs in plants compared to animals, fungi and prokaryotes. The plant SDRs were first classified into three major types --- 'classical', 'extended' and 'divergent' --- but a minority (10 % of the predicted SDRs) could not be classified into these general types ('unknown' or 'atypical' types). In a second step, we could categorize the vast majority of land plant SDRs into a set of 49 families. Out of these 49 families, 35 appeared early during evolution since they are commonly found through all the Green Lineage. Yet, some SDR families --- tropinone reductase-like proteins (SDR65C), 'ABA2-like'-NAD dehydrogenase (SDR110C), 'salutaridine/menthone-reductase-like' proteins (SDR114C), 'dihydroflavonol 4-reductase'-like proteins (SDR108E) and 'isoflavone-reductase-like' (SDR460A) proteins --- have undergone significant functional diversification within vascular plants since they diverged from Bryophytes. Interestingly, these diversified families are either involved in the secondary metabolism routes (terpenoids, alkaloids, phenolics) or participate in developmental processes (hormone biosynthesis or catabolism, flower development), in opposition to SDR families involved in primary metabolism which are poorly diversified. Conclusion The application of HMMs to plant genomes enabled us to identify 49 families that encompass all Angiosperms ('higher plants') SDRs, each family being sufficiently conserved to enable simpler analyses based only on overall sequence similarity. The multiplicity of SDRs in plant kingdom is mainly explained by the diversification of large families involved in different secondary metabolism pathways, suggesting that the chemical diversification that accompanied the emergence of vascular plants acted as a driving force for SDR evolution