Project description:miR-30d has been identified in this study as a novel onco-miRNA downstream of mutant p53. Here we report the microarray data obtained in MDA-MB-231 in which miR-30d levels and function were stably inhibited by a decoy construct (dy_30d)
Project description:Golgi biogenesis is a crucial cellular process, yet its study has been hindered by the lack of easy methods to induce massive production of new Golgi units. In this study, we optimized an enzyme inactivation approach to stimulate the formation of fully functional Golgi stacks. Massive Golgi biogenesis was achieved through DAB-mediated inactivation of preexisting Golgi structures in cells stably expressing the chimeric Golgi enzyme Mannosidase II fused with horse radish peroxidase (HRP). Within 10 to 18 hours post-Golgi inactivation, substantial number of cells regenerated new Golgi stacks capable of transporting various cargo proteins to their appropriate destinations. This functional and structural recovery of the Golgi in ManII-HRP cells offers an efficient system to study Golgi biogenesis and associated cellular responses. We further explored transcriptomic changes in ManII-HRP cells, discovering that the extensive biogenesis of Golgi membranes induces the transcriptional activation of numerous Golgi-associated genes. These genes encode proteins involved in diverse functions such as glycosylation, membrane tethering, and ion/sugar transport. The timing of this gene transactivation correlates with the restoration of Golgi cisternae architecture and trafficking capabilities, suggesting that this transcriptional response plays an integral role in the biogenesis of new Golgi units.
Project description:The process of aging is associated with disturbed mineral homeostasis, such as zinc deficiency. Simultaneously, cellular response to external stimuli, including receptor signaling and DNA repair response diminishes, and epigenetic dysregulation occurs. The Golgi apparatus plays various roles in the cell; however, the effect of aging on the morphology and function of the Golgi apparatus and the impact of Golgi senescence in cellular activity remains unknown. In the present study, we found that Golgi stress is associated with senescent cell irresponsiveness to external stimuli. The senescent Golgi was associated with a disassembled microtubule network in the Golgi and perinuclear areas. These effects could be reproduced by disruption of the Golgi-associated microtubules or zinc depletion. To clarify the importance of Golgi-zinc homeostasis in more detail, the implications of the Golgi-zinc transporter ZIP13 were analyzed; Zip13-deficient mice Golgi exhibited morphology similar to that of senescent Golgi. Additionally, fibroblasts from Zip13-deficient mice showed dysregulation of DNA repair and cellular acetylation with downregulated nuclear translocation of p300, HDAC1/2, and p53 proteins, which are reminiscent characteristics of senescent cells. Our findings demonstrate the underlying reason for senescent cell irresponsiveness to various stimuli and highlight the importance of a zinc-rich diet during aging.
Project description:Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post59 translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In both cancer cell cultures and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycomes revealed that fucosylation of the antioxidant PON1 is a critical component of the therapy66 induced secretome. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Non-specific and PON1-specific secretome deglycosylation both limited the expansion of resistant clones in a tumor regression model. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.
Project description:TGFβ ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. We now show that TGFβ-dependent cell migration, invasion and metastasis are empowered by mutant-p53. To investigate the specific gene expression program by which mutant-p53 and TGFβ control invasion and metastasis in breast cancer cells, we compared the TGFβ transcriptomic profile of control and mutant-p53 depleted MDA-MB-231 cells. Keywords: expression profiling by array
Project description:Secretome is intimately linked to Golgi apparatus (GA) which acts as a transition platform of proteins processing and sorting in trafficking pathways. GA-associated secretome runs through the endomembrane system and is critical for inter- and intracellular communication networks. However, achieving in situ dissection of GA-associated secretome remains challenging owing to the lack of efficient labeling methods. Herein, we described an aggregation-induced emission luminogen-mediated photocatalytic proximity labeling strategy (AIE-PhoPL) that allowed dynamic profiling of GA-associated secretome with high spatiotemporal precision in minutes. With the GA-targeting photocatalyst based on aggregation-induced emission luminogen (AIEgen), the proteins were labeled locally in Golgi lumen with high efficiency under light, enabling spatiotemporally resolved modifying of histidine and tyrosine residues. AIE-PhoPL strategy was successfully utilized in living HeLa and hard-to-transfect macrophage HMC3 cells by determining a significant subset of GA-associated secretome with 80% specificity, linking the distinct GA-associated secretory profiles to cellular characteristics. We further applied AIE-PhoPL to dynamic proteome mapping of brain and bone metastatic lung cancer cells, which revealed GA-associated secretory proteomic patterns related to metastatic mechanisms and the underlying roles GA-associated secretion played in extracellular matrix organization during metastasis. This work provides a powerful tool to break out the dilemma for chemical labeling of GA-associated secretome in living cells and fuels the research on diversification of controllable chemical probes for subcellular proteome labeling.
Project description:We explore the transcriptional response of mammalian cells undergoing various insults to Golgi homeostasis. HEK293 cells (Flp-In T-REx 293 cells) stably containing a doxycycline-inducible Golgi-localized HaloTag2 construct (GA-HT2) were treated with the ionophore nigericin, the glycosylation inhibitor xyloside, or were induced by doxycycline and treated with the hydrophobic tag HyT36 to induce destabilization of GA-HT2. We found that while nigericin and xyloside induce global transcriptional changes, destabilization of GA-HT2 induces a Golgi-specific response.