MAGOH and MAGOHB Knockdown in Melanoma Cells Decreases Nonsense-Mediated Decay Activity and Promotes Apoptosis via Upregulation of GADD45A.

Cutaneous malignant melanoma is a highly proliferative and aggressive skin cancer with a steadily increasing incidence and a low long-term survival rate after metastatic progression. The protein MAGOH and its highly identical homologue MAGOHB are core components of the exon junction complex (EJC), which regulates splicing, stability and translation of mRNAs. The EJC, and especially MAGOH, has been shown to be involved in the development and progression of several cancers. In melanoma, the expression and function of both homologues remain essentially unexplored. This study identifies high MAGOH and MAGOHB protein expression in cutaneous melanoma cell lines and patient derived tissue samples. An siRNA-mediated knockdown of MAGOH significantly inhibits melanoma cell proliferation. The loss of MAGOH does not affect cell cycle progression, but induces apoptosis, an effect that is enhanced by a simultaneous knockdown of MAGOH and MAGOHB. MAGOH and MAGOHB do not influence the expression of the pro-apoptotic protein Bcl-XS or exon skipping. However, the knockdown of MAGOH and MAGOHB strongly decreases nonsense-mediated decay (NMD) activity, leading to an upregulation of the pro-apoptotic protein GADD45A. In conclusion, simultaneous inhibition of MAGOH and MAGOHB expression substantially affects cell survival, indicating both MAGOH homologues as promising new targets for the treatment of melanoma.

A previously unknown Argonaute 2 variant positively modulates the viability of melanoma cells.

In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.

The Putative Bromodomain Protein PfBDP7 of the Human Malaria Parasite Plasmodium Falciparum Cooperates With PfBDP1 in the Silencing of Variant Surface Antigen Expression.

Epigenetic regulation is a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite Plasmodium (P.) falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the P. falciparum bromodomain proteins (PfBDP) 1 and PfBDP2 (BDP1/BDP2 core complex) was previously shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7, because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, STEVOR, and PfMC-2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and established PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin.

Due to the increasing development of antibiotic resistances in recent years, scientists search intensely for new methods to control bacteria. Photodynamic treatment with porphyrins such as chlorophyll derivatives is one of the most promising methods to handle bacterial infestation, but their use is dependent on illumination and they seem to be more effective against Gram-positive bacteria than against Gram-negatives. In this study, we tested chlorophyllin against three bacterial model strains, the Gram-positive Bacillus subtilis 168, the Gram-negative Escherichia coli DH5α and E. coli strain NR698 which has a deficient outer membrane, simulating a Gram-negative "without" its outer membrane. Illuminated with a standardized light intensity of 12 mW/cm², B. subtilis showed high sensitivity already at low chlorophyllin concentrations (≤105 cfu/mL: ≤0.1 mg/L, 106⁻108 cfu/mL: 0.5 mg/L), whereas E. coli DH5α was less sensitive (≤105 cfu/mL: 2.5 mg/L, 106 cfu/mL: 5 mg/L, 107⁻108 cfu/mL: ineffective at ≤25 mg/L chlorophyllin). E. coli NR698 was almost as sensitive as B. subtilis against chlorophyllin, pointing out that the outer membrane plays a significant role in protection against photodynamic chlorophyllin impacts. Interestingly, E. coli NR698 and B. subtilis can also be inactivated by chlorophyllin in darkness, indicating a second, light-independent mode of action. Thus, chlorophyllin seems to be more than a photosensitizer, and a promising substance for the control of bacteria, which deserves further investigation.