RESUMO
In this work, we are reporting that "Shock and Kill", a therapeutic approach designed to eliminate latent HIV from cell reservoirs, is extrapolatable to cancer therapy. This is based on the observation that malignant cells express a spectrum of human endogenous retroviral elements (HERVs) which can be transcriptionally boosted by HDAC inhibitors. The endoretroviral gene HERV-V2 codes for an envelope protein, which resembles syncytins. It is significantly overexpressed upon exposure to HDAC inhibitors and can be effectively targeted by simultaneous application of TLR7/8 agonists, triggering intrinsic apoptosis. We demonstrated that this synergistic cytotoxic effect was accompanied by the functional disruption of the TLR7/8-NFκB, Akt/PKB, and Ras-MEK-ERK signalling pathways. CRISPR/Cas9 ablation of TLR7 and HERV-V1/V2 curtailed apoptosis significantly, proving the pivotal role of these elements in driving cell death. The effectiveness of this new approach was confirmed in ovarian tumour xenograft studies, revealing a promising avenue for future cancer therapies.
Assuntos
Adjuvantes Imunológicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Retrovirus Endógenos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptor 7 Toll-Like/agonistas , Ativação Viral/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Depsipeptídeos/farmacologia , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Feminino , Humanos , Imiquimode/farmacologia , Imunidade Inata/efeitos dos fármacos , Camundongos Nus , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/virologia , Pteridinas/farmacologia , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Células Tumorais Cultivadas , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vorinostat/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human apolipoprotein D (ApoD) occurs in plasma associated with high density lipoprotein. Apart from the involvement in lipid metabolism, its binding activity for progesterone and arachidonic acid plays a role in cancer development and neurological diseases. The crystal structures of free ApoD and its complex with progesterone were determined at 1.8A resolution and reveal a lipocalin fold. The narrow, mainly uncharged pocket within the typical beta-barrel accommodates progesterone with its acetyl side chain oriented toward the bottom. The cavity adopts essentially the same shape in the absence of progesterone and allows complexation of arachidonic acid as another cognate ligand. Three of the four extended loops at the open end of the beta-barrel expose hydrophobic side chains, which is an unusual feature for lipocalins and probably effects association with the high density lipoprotein particle by mediating insertion into the lipid phase. This mechanism is in line with an unpaired Cys residue in the same surface region that can form a disulfide cross-link with apolipoprotein A-II.
Assuntos
Apolipoproteínas D/química , Metabolismo dos Lipídeos , Lipoproteínas HDL/química , Progesterona/química , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Apolipoproteínas D/metabolismo , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Lipoproteínas HDL/metabolismo , Neoplasias/metabolismo , Doenças do Sistema Nervoso/metabolismo , Progesterona/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Human apolipoprotein D (ApoD) is a physiologically important member of the lipocalin protein family that was discovered as a peripheral subunit of the high-density lipoprotein (HDL) but is also abundant in other body fluids and organs, including neuronal tissue. Although it has been possible to produce functional ApoD in the periplasm of Escherichia coli and to demonstrate its ligand-binding activity for progesterone and arachidonic acid, the recombinant protein suffers from a pronounced tendency to aggregate and to adsorb to vessel surfaces as well as chromatography matrices, thus hampering further structural investigation. Here, we describe a systematic mutagenesis study directed at presumably exposed hydrophobic side chains of the unglycosylated recombinant protein. As a result, one ApoD mutant with just three new amino acid substitutions--W99H, I118S, and L120S--was identified, which exhibits the following features: (1) improved yield upon periplasmic biosynthesis in E. coli, (2) elution as a monomeric protein from a gel permeation chromatography column, and (3) unchanged binding activity for its physiological ligands. In addition, the engineered ApoD was successfully crystallized (space group I4 with unit cell parameters a = 75.1 A, b = 75.1 A, c = 166.0 A, alpha = beta = gamma = 90 degrees), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein.