Biology subjects

Zaver, S. A.

Publications and source records attributed to Zaver, S. A..

5 recordsLinked to original sources

bioRxiv2022

STING activation depends on ACBD3 and other phosphatidylinositol 4-phosphate-regulating proteins

STING induces transcription of pro-inflammatory genes upon activation at the Golgi apparatus. Many of the regulators involved in STING activation are unknown. We found that ACBD3 and other phosphatidylinositol 4-phosohate (PI4P) regulating proteins play a critical role in STING activation. We show that proper STING localization and activation at the Golgi depended on ACBD3 and PI4KB expression. Furthermore, depleting PI4P by inactivating PI4KB or overexpressing Sac1 diminished STING activation. STING signalling was also regulated by the lipid-shuttling protein OSBP, which removes PI4P from the Golgi. OSBP inhibition by the FDA-approved antifungal itraconazole and other OSBP inhibitors greatly enhanced STING activation by increasing the levels of STING-activating phospholipids. Itraconazole-enhanced STING activation resulted in a hundred to thousand-fold increased expression of interferon-beta and other cytokines. In conclusion, the phospholipid PI4P is critical for STING activation and manipulating PI4P levels is a promising therapeutic strategy to alter the STING immune response.

immunology↗

bioRxiv2022

The proto-oncogene c-Src phosphorylates cGAS to reduce DNA binding and activation

cGMP-AMP synthase (cGAS) is a DNA sensor and responsible for inducing an anti-tumor immune response. Recent studies reveal cGAS is frequently inhibited in cancer, and therapeutic targets to promote anti-tumor cGAS function remain elusive. c-Src is a proto-oncogene tyrosine kinase and is expressed at elevated levels in numerous cancers. Here, we demonstrate that c-Src expression in tumors from various cancers negatively correlates with innate immune gene expression and patient survival. We determine that c-Src restricts cGAS signaling in human cell lines through c-Src small molecule inhibitors, depletion, and overexpression. cGAS and c-Src interact in cells and in vitro, while c-Src directly inhibits cGAS enzymatic activity and DNA binding in a kinase-dependent manner. c-Src phosphorylates cGAS, and inhibition of cGAS Y248 phosphorylation partially reduces c-Src inhibition. Collectively, our study demonstrates that cGAS anti-tumor signaling is hindered by the proto-oncogene c-Src and describes how cancer-associated proteins can regulate the innate immune system.

molecular biology↗

bioRxiv2021

Sequence diversity in the 3' untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

Alphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3 untranslated region (3UTR) of the virus. We investigated the potential role of variability in the 3UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

microbiology↗

bioRxiv2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

immunology↗

bioRxiv2021

A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics

33-cyclic di-adenosine monophosphate (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. C-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure guided design of a Forster resonance energy transfer (FRET) based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows for the detection of c-di-AMP dynamics both in vitro and within live single cells in a nondestructive manner. Our initial studies identify a unimodal distribution of c-di-AMP in Bacillus subtilis which decreases rapidly when cells are grown in diluted Luria Broth. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses, again in a unimodal manner. These findings provide novel insight into c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. ImportanceC-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineer and implement a c-di-AMP specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.

bioengineering↗