Cell survival and protein secretion associated with Golgi integrity in response to Golgi stress-inducing agents.

The Golgi apparatus is part of the secretory pathway and of central importance for modification, transport and sorting of proteins and lipids. ADP-ribosylation factors, whose activation can be blocked by brefeldin A (BFA), play a major role in functioning of the Golgi network and regulation of membrane traffic and are also involved in proliferation and migration of cancer cells. Due to high cytotoxicity and poor bioavailability, BFA has not passed the preclinical stage of drug development. Recently, AMF-26 and golgicide A have been described as novel inhibitors of the Golgi system with antitumor or bactericidal properties. We provide here further evidence that AMF-26 closely mirrors the mode of action of BFA but is less potent. Using several human cancer cell lines, we studied the effects of AMF-26, BFA and golgicide A on cell homeostasis including Golgi structure, endoplasmic reticulum (ER) stress markers, secretion and viability, and found overall a significant correlation between these parameters. Furthermore, modulation of ADP-ribosylation factor expression has a profound impact on Golgi organization and survival in response to Golgi stress inducers.

TRAPPC13 modulates autophagy and the response to Golgi stress.

Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium Shigella flexneri in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.

SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB.

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to long-lasting lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses, which can cause systemic complications. Here, we have evaluated transcriptional and cytokine secretion profiles and detected a distinct upregulation of inflammatory cytokines in infected cell cultures and samples taken from infected patients. Building on these observations, we found a specific activation of NF-κB and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-κB response was mediated by cGAS-STING activation and could be attenuated through several STING-targeting drugs. Our results show that SARS-CoV-2 directs a cGAS-STING mediated, NF-κB-driven inflammatory immune response in human epithelial cells that likely contributes to inflammatory responses seen in patients and could be therapeutically targeted to suppress severe disease symptoms.

Golgi stress mediates redox imbalance and ferroptosis in human cells.

Cytotoxic activities of several Golgi-dispersing compounds including AMF-26/M-COPA, brefeldin A and golgicide A have previously been shown to induce autophagy or apoptosis. Here, we demonstrate that these Golgi disruptors also trigger ferroptosis, a non-apoptotic form of cell death characterized by iron-dependent oxidative degradation of lipids. Inhibitors of ferroptosis not only counteract cell death, but they also protect from Golgi dispersal and inhibition of protein secretion in response to several Golgi stress agents. Furthermore, the application of sublethal doses of ferroptosis-inducers such as erastin and sorafenib, low cystine growth conditions, or genetic knockdown of SLC7A11 and GPX4 all similarly protect cells from Golgi stress and lead to modulation of ACSL4, SLC7A5, SLC7A11 or GPX4 levels. Collectively, this study suggests a previously unrecognized function of the Golgi apparatus, which involves cellular redox control and prevents ferroptotic cell death.

Golgi stress-induced transcriptional changes mediated by MAPK signaling and three ETS transcription factors regulate MCL1 splicing.

The secretory pathway is a major determinant of cellular homoeostasis. While research into secretory stress signaling has so far mostly focused on the endoplasmic reticulum (ER), emerging data suggest that the Golgi itself serves as an important signaling hub capable of initiating stress responses. To systematically identify novel Golgi stress mediators, we performed a transcriptomic analysis of cells exposed to three different pharmacological compounds known to elicit Golgi fragmentation: brefeldin A, golgicide A, and monensin. Subsequent gene-set enrichment analysis revealed a significant contribution of the ETS family transcription factors ELK1, GABPA/B, and ETS1 to the control of gene expression following compound treatment. Induction of Golgi stress leads to a late activation of the ETS upstream kinases MEK1/2 and ERK1/2, resulting in enhanced ETS factor activity and the transcription of ETS family target genes related to spliceosome function and cell death induction via alternate MCL1 splicing. Further genetic analyses using loss-of-function and gain-of-function experiments suggest that these transcription factors operate in parallel.

Image-based drug screen identifies HDAC inhibitors as novel Golgi disruptors synergizing with JQ1.

The Golgi apparatus is increasingly recognized as a major hub for cellular signaling and is involved in numerous pathologies, including neurodegenerative diseases and cancer. The study of Golgi stress-induced signaling pathways relies on the selectivity of the available tool compounds of which currently only a few are known. To discover novel Golgi-fragmenting agents, transcriptomic profiles of cells treated with brefeldin A, golgicide A, or monensin were generated and compared with a database of gene expression profiles from cells treated with other bioactive small molecules. In parallel, a phenotypic screen was performed for compounds that alter normal Golgi structure. Histone deacetylase (HDAC) inhibitors and DNA-damaging agents were identified as novel Golgi disruptors. Further analysis identified HDAC1/HDAC9 as well as BRD8 and DNA-PK as important regulators of Golgi breakdown mediated by HDAC inhibition. We provide evidence that combinatorial HDACi/(+)-JQ1 treatment spurs synergistic Golgi dispersal in several cancer cell lines, pinpointing a possible link between drug-induced toxicity and Golgi morphology alterations.