Transcriptomics Reveals the Mevalonate and Cholesterol Pathways Blocking as Part of the Bacterial Cyclodipeptides Cytotoxic Effects in HeLa Cells of Human Cervix Adenocarcinoma.

The incidence of human cervix adenocarcinoma (CC) caused by papillomavirus genome integration into the host chromosome is the third most common cancer among women. Bacterial cyclodipeptides (CDPs) exert cytotoxic effects in human cervical cancer HeLa cells, primarily by blocking the PI3K/Akt/mTOR pathway, but downstream responses comprising gene expression remain unstudied. Seeking to understand the cytotoxic and anti-proliferative effects of CDPs in HeLa cells, a global RNA-Seq analysis was performed. This strategy permitted the identification of 151 differentially expressed genes (DEGs), which were either up- or down-regulated in response to CDPs exposure. Database analysis, including Gene Ontology (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed differential gene expression on cancer transduction signals, and metabolic pathways, for which, expression profiles were modified by the CDPs exposure. Bioinformatics confirmed the impact of CDPs in the differential expression of genes from signal transduction pathways such as PI3K-Akt, mTOR, FoxO, Wnt, MAPK, P53, TGF-ß, Notch, apoptosis, EMT, and CSC. Additionally, the CDPs exposure modified the expression of cancer-related transcription factors involved in the regulation of processes such as epigenetics, DNA splicing, and damage response. Interestingly, transcriptomic analysis revealed the participation of genes of the mevalonate and cholesterol biosynthesis pathways; in agreement with this observation, total cholesterol diminished, confirming the blockage of the cholesterol synthesis by the exposure of HeLa cells to CDPs. Interestingly, the expression of some genes of the mevalonate and cholesterol synthesis such as HMGS1, HMGCR, IDI1, SQLE, MSMO1, SREBF1, and SOAT1 was up-regulated by CDPs exposure. Accordingly, metabolites of the mevalonate pathway were accumulated in cultures treated with CDPs. This finding further suggests that the metabolism of cholesterol is crucial for the occurrence of CC, and the blockade of the sterol synthesis as an anti-proliferative mechanism of the bacterial CDPs, represents a reasonable chemotherapeutic drug target to explore. Our transcriptomic study supports the anti-neoplastic effects of bacterial CDPs in HeLa cells shown previously, providing new insights into the transduction signals, transcription factors and metabolic pathways, such as mevalonate and cholesterol that are impacted by the CDPs and highlights its potential as anti-neoplastic drugs.

MEDIATOR18 influences Arabidopsis root architecture, represses auxin signaling and is a critical factor for cell viability in root meristems.

The Mediator (MED) complex plays a key role in the recruitment and assembly of the transcription machinery for the control of gene expression. Here, we report on the role of MEDIATOR18 (MED18) subunit in root development, auxin signaling and meristem cell viability in Arabidopsis thaliana seedlings. Loss-of-function mutations in MED18 reduce primary root growth, but increase lateral root formation and root hair development. This phenotype correlates with alterations in cell division and elongation likely caused by an increased auxin response and transport at the root tip, as evidenced by DR5:GFP, pPIN1::PIN1-GFP, pPIN2::PIN2-GFP and pPIN3::PIN3-GFP auxin-related gene expression. Noteworthy, med18 seedlings manifest cell death in the root meristem, which exacerbates with age and/or exposition to DNA-damaging agents, and display high expression of the cell regeneration factor ERF115. Cell death in the root tip was reduced in med18 seedlings grown in darkness, but remained when only the shoot was exposed to light, suggesting that MED18 acts to protect root meristem cells from local cell death, and/or in response to root-acting signal(s) emitted by the shoot in response to light stimuli. These data point to MED18 as an important component for auxin-regulated root development, cell death and cell regeneration in root meristems.

Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus: An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress.

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

Alkamides activate jasmonic acid biosynthesis and signaling pathways and confer resistance to Botrytis cinerea in Arabidopsis thaliana.

Alkamides are fatty acid amides of wide distribution in plants, structurally related to N-acyl-L-homoserine lactones (AHLs) from Gram-negative bacteria and to N- acylethanolamines (NAEs) from plants and mammals. Global analysis of gene expression changes in Arabidopsis thaliana in response to N-isobutyl decanamide, the most highly active alkamide identified to date, revealed an overrepresentation of defense-responsive transcriptional networks. In particular, genes encoding enzymes for jasmonic acid (JA) biosynthesis increased their expression, which occurred in parallel with JA, nitric oxide (NO) and H2O2 accumulation. The activity of the alkamide to confer resistance against the necrotizing fungus Botrytis cinerea was tested by inoculating Arabidopsis detached leaves with conidiospores and evaluating disease symptoms and fungal proliferation. N-isobutyl decanamide application significantly reduced necrosis caused by the pathogen and inhibited fungal proliferation. Arabidopsis mutants jar1 and coi1 altered in JA signaling and a MAP kinase mutant (mpk6), unlike salicylic acid- (SA) related mutant eds16/sid2-1, were unable to defend from fungal attack even when N-isobutyl decanamide was supplied, indicating that alkamides could modulate some necrotrophic-associated defense responses through JA-dependent and MPK6-regulated signaling pathways. Our results suggest a role of alkamides in plant immunity induction.