RESUMO
Microvesicles (MVs) containing proteins, nucleic acid or organelles are shed from the plasma membrane. Although the mechanisms of MV budding are well elucidated, the connection between endosomal trafficking and MV formation remains poorly understood. In this report, RAB22A is revealed to be crucial for EGFR-containing MVs formation by the RAB GTPase family screening. RAB22A recruits TBC1D2B, a GTPase-activating protein (GAP) of RAB7A, to inactivate RAB7A, thus preventing EGFR from being transported to late endosomes and lysosomes. RAB22A also engages SH3BP5L, a guanine-nucleotide exchange factor (GEF) of RAB11A, to activate RAB11A on early endosomes. Consequently, EGFR is recycled to the cell surface and packaged into MVs. Furthermore, EGFR can phosphorylate RAB22A at Tyr136, which in turn promotes EGFR-containing MVs formation. Our findings illustrate that RAB22A acts as a sorter on early endosomes to sort EGFR to recycling endosomes for MV shedding by both activating RAB11A and inactivating RAB7A.
Assuntos
Endossomos , Receptores ErbB , Proteínas rab de Ligação ao GTP , Receptores ErbB/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Humanos , Transporte Proteico , Micropartículas Derivadas de Células/metabolismo , proteínas de unión al GTP Rab7/metabolismo , Células HeLa , Proteínas Ativadoras de GTPase/metabolismo , Lisossomos/metabolismoRESUMO
Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a non-genotoxic rat carcinogen, which induced mammary tumors in male and female rats in a two-year bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 weeks. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary and liver samples using ultra high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as non-genotoxic carcinogen. Mammary DNA samples (n = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations (CDMs), namely regions of Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, p < 0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 weeks as compared to 12 weeks. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a non-genotoxic carcinogen using a treatment duration as short as 3 months.
RESUMO
BACKGROUND: Vesicular stomatitis virus (VSV) is a typical non-segmented negative-sense RNA virus of the genus Vesiculovirus in the family Rhabdoviridae. VSV can infect a wide range of animals, including humans, with oral blister epithelial lesions. VSV is an excellent model virus with a wide range of applications as a molecular tool, a vaccine vector, and an oncolytic vector. To further understand the interaction between VSV and host cells and to provide a theoretical basis for the application prospects of VSV, we analyzed the expression of host differentially expressed genes (DEGs) during VSV infection using RNA-Seq. RESULTS: Our analyses found a total of 1015 differentially expressed mRNAs and 161 differentially expressed LncRNAs in BHK-21 cells infected with VSV for 24 h compared with controls. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment showed that the differentially expressed lncRNAs and their target genes were mainly concentrated in pathways related to apoptosis, cancer, disease, and immune system activation, including the TNF, P53, MAPK, and NF-kappaB signaling pathways. The differentially expressed lncRNA can modulate immune processes by regulating genes involved in these signaling transmissions. Ten randomly selected DEGs, namely, Il12rb2, F2, Masp2, Mcl1, FGF18, Ripk1, Fas, BMF, POLK, and JAG1, were validated using RT-qPCR. As predicted through RNA-Seq analysis, these DEGs underwent either up- or downregulation, suggesting that they may play key regulatory roles in the pathways mentioned previously. CONCLUSIONS: Our study showed that VSV infection alters the host metabolic network and activates immune-related pathways, such as MAPK and TNF. The above findings provide unique insights for further study of the mechanism of VSV-host interactions and, more importantly, provide a theoretical basis for VSV as an excellent vaccine carrier.