No abstract

Stimulator of interferon genes (STING) is an intracellular sensor of cyclic di-nucleotides involved in the innate immune response against pathogen- or self-derived DNA. STING trafficking is tightly linked to its function, and its dysregulation can lead to disease. Here, we systematically characterize genes regulating STING trafficking and examine their impact on STING-mediated responses. Using proximity-ligation proteomics and genetic screens, we demonstrate that an endosomal sorting complex required for transport (ESCRT) complex containing HGS, VPS37A and UBAP1 promotes STING degradation, thereby terminating STING-mediated signaling. Mechanistically, STING oligomerization increases its ubiquitination by UBE2N, forming a platform for ESCRT recruitment at the endosome that terminates STING signaling via sorting in the lysosome. Finally, we show that expression of a UBAP1 mutant identified in patients with hereditary spastic paraplegia and associated with disrupted ESCRT function, increases steady-state STING-dependent type I IFN responses in healthy primary monocyte-derived dendritic cells and fibroblasts. Based on these findings, we propose that STING is subject to a tonic degradative flux and that the ESCRT complex acts as a homeostatic regulator of STING signaling.

While gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of “connectivity” to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics.

STING is an intracellular sensor of cyclic di-nucleotides involved in response to pathogen- or self-derived DNA that induces protective immunity, or if dysregulated, autoimmunity. STING trafficking is tightly linked to its activity. We aimed to systematically characterize genes regulating STING trafficking and to define their impact on STING responses. Based on proximity-ligation proteomics and genetic screens, an ESCRT complex containing HGS, VPS37A and UBAP1 was found to be required for STING degradation and signaling shutdown. Analogous to phosphorylated STING creating a platform for IRF3 recruitment, oligomerization-driven STING ubiquitination by UBE2N formed a platform for ESCRT recruitment at the endosome, responsible for STING signaling shutdown. A UBAP1 mutant that underlies human spastic paraplegia and disrupts ESCRT function led to STING-dependent type I IFN responses at the steady-state, defining ESCRT as a homeostatic regulator of STING signaling.

Parkinson's disease (PD) is a fatal, common, neurodegenerative disorder that affects up to 10 million people worldwide, and lacks disease altering treatment or prevention. It is the second most common neurodegenerative disease globally and is characterized by loss of dopaminergic neurons initially in the substantia nigra. Epidemiological studies have shown that patients with PD have a higher risk of developing melanoma but a lower risk of developing other cancers. According to published reports, the co‐occurrence of melanoma and PD and vice versa is much higher than expected with an odds ratio up to 6 fold. Interestingly, the risk factors of developing PD increase with a family history of melanoma, lighter skin and hair color. Patients who have a first degree relative with either disease also have an increased risk to develop the other disease. The aim of this project was to study the mechanism of association between PD and melanoma. Melanoma cells (SK‐MEL2) that overexpress wild‐type a‐synuclein (the CNS‐specific protein known to aggregate in the brains of Parkinson's patients) and mutant a‐synuclein (A53T, a point mutation that causes autosomal dominant, inherited Parkinson's disease) were used as a model. Menandione was used to induce oxidative stress in the cells, and expression of a‐synuclein and melanin in these cells by Western Blotting. Immunochemistry was utilized to identify co‐localization of a‐synuclein and melanin in the Golgi and melanosomes. This indicates there are potentially common signaling pathway that could explain the comorbidity between the diseases. In addition, we also report the presence of cytoplasmic a‐synuclein and melanin in the cells, implying a possible common secretion pathways for the two proteins. Our observation of localized a‐synuclein in the lysosomes, ensures future areas of potential importance in the interaction and mechanism of lysosomes and a‐synuclein dissection. Additional testing with other cancer cell lines needs to be carried out to ensure that this observed pattern is seen only in melanoma.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.