RESUMO
Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 µM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by â¼50-60%, heparan sulfate by â¼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by â¼74 and â¼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by â¼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.
Assuntos
Acetilgalactosamina/análogos & derivados , Glicosaminoglicanos/biossíntese , Acetilgalactosamina/química , Acetilgalactosamina/farmacologia , Animais , Linhagem Celular , Embrião de Galinha , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glicosaminoglicanos/antagonistas & inibidores , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Polissacarídeos/antagonistas & inibidores , Polissacarídeos/biossíntese , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR-Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference-mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.