Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, in which most viral genes are silenced except a few latent proteins such as latency-associated nuclear antigen (LANA). In latent viral chromatin, viral genes are poised to be transcribed, and KSHV LANA plays a major role in maintaining such transcription status. In the poised chromatins, LANA recruits cellular CHD4 (Chromodomain Helicase DNA binding protein 4) and suppresses inducible viral gene promoters. The CHD4 is known to regulate cell differentiation by restricting enhancer-promoter interactions and its mutation or overexpression deregulates the host cell transcription program and therefore associates with tumorigenesis. Here, we identified a putative CHD4 inhibitor from the LANA amino acid sequence from the LANA-CHD4 interaction surface. The small peptide interacts with CHD4 at its PHD domain with 14 nM KD (dissociation constant). The introduction of the peptide into the primary effusion lymphomas induces caspase-mediated CHD4 cleavage and subsequently triggered cell apoptosis and autophagy. A series of MTT assays demonstrated that the peptide preferentially killed lymphoma and leukemia cell lines at low micromolar concentrations, while peripheral mononuclear cells or adhesion cell lines were found to be more resistant to the peptide treatment. A monocyte cell differentiation model demonstrated that pre-treatment with the peptide at a low dose substantially enhanced transitioning into M2 macrophage (by reducing CHD4 occupancies from gene promoters), and globally altered the repertories of phorbol myristate acetate target genes in U937 cells. Finally, the PEL xenograft mouse model inhibited tumor growth without measurable side effects, and PEL cells isolated from xenograft tumors showed reduced CHD4 and LANA expression with terminally differentiated phenotype. We propose that the peptide isolated from the KSHV LANA sequence is biologically active and may function as a CHD4 inhibitor.

Project description:ERM-NM-117p is a synthetic peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERM-NM-1) and initially synthesized to mimic its calmodulin binding site. ERM-NM-117p was subsequently found to elicit estrogenic responses in E2-deprived ERM-NM-1-positive breast cancer cells, increasing proliferation and E2-dependent gene transcription. Surprisingly, in E2-supplemented media, ERM-NM-117p induced apoptosis and modified the actin network, influencing thereby cell motility. Here, we report that ERM-NM-117p induces a massive early (3h) transcriptional activity in breast cancer cell lines SKBR3). Remarkably, about 75% of the significantly modified transcripts were also modified by E2, confirming the pro-estrogenic profile of ERM-NM-117p. The different ER spectra of the used cell lines allowed us to extract a specific ERM-NM-117p signature related to ERM-NM-1 and its variant ERM-NM-136. With respect to ERM-NM-1, the peptide activates nuclear (cell cycle, cell proliferation, nucleic acid and protein synthesis) and extranuclear signaling pathways. In contrast, through ERM-NM-136 it exerts inhibitory events on inflammation and cell cycle and inhibition of EGFR signaling. This is the first work reporting ERM-NM-136 specific transcriptional effects. The fact that a number ERM-NM-117p-induced transcripts is different from those activated by E2 revealed that the apoptosis and actin modifying effects of ERM-NM-117p are independent from the ER-related actions of the peptide. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2 (10-6M) or ERa17p in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturerM-bM-^@M-^Ys instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.

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Peptide barcoding for one-pot evaluation of sequence-function relationships of free nanobodies

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