Browsing by Author "Paul, Swagatika"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- NAK-associated protein 1/NAP1 activates TBK1 to ensure accurate mitosis and cytokinesisPaul, Swagatika; Sarraf, Shireen; Nam, K. H.; Zavar, Leila; DeFoor, Nicole; Biswas, Sahitya; Fritsch, Lauren; Yaron, Tomer; Johnson, Jared; Huntsman, Emily; Cantley, Lewis; Ordureau, Alban; Pickrell, Alicia M. (Rockefeller University Press, 2023-12-07)Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.
- The Non-canonical Function and Regulation of TBK1 in the Cell CyclePaul, Swagatika (Virginia Tech, 2023-10-11)Protein kinases play essential roles in orchestrating almost every step during mitosis. Aberrant kinase activity often leads to errors in the cell cycle progression which consequently becomes the underlying cause for developmental defects or abnormal cell proliferation leading to cancer. Tank Binding Kinase 1 (TBK1) is overexpressed in certain cancer types and is activated on the centrosomes during mitosis. Loss of TBK1 impairs cell division resulting in growth defects and the accumulation of multinucleated cells. Therefore, proper activation and localization of TBK1 are essential for mitotic progression. Yet, the upstream regulation of TBK1 and the function of activated TBK1 on the centrosomes is unknown. Also, the cause and consequences of overexpression of TBK1 in cancers remain to be explored. Activation of TBK1 depends on its binding to an adaptor protein which induces a conformational change leading to trans autophoshorylation on serine 172 of its kinase domain. We identified that an established innate immune response protein, NAK Associated Protein1 (NAP1/AZI2), is the adaptor required for binding and activating TBK1 during mitosis. Loss of either NAP1 or TBK1 results in the accumulation of binucleated and multinucleated cells, possibly due to several mitotic and cytokinetic defects seen in these knockout (KO) cells. We establish NAP1 as a cell cycle regulated protein which colocalizes with activated TBK1 on the centrosomes during mitosis. Furthermore, by performing an unbiased quantitative phosphoproteomics analysis during mitosis, the substrates discovered reveal that TBK1 also regulates other known cell cycle regulating kinases such as Aurora A and Aurora B. TBK1 is also an established autophagy protein and since the autophagy machinery is often impaired or remodeled to facilitate rapid cell division, we evaluated the underlying connection between TBK1 activation and autophagy. The data shows that cells lacking the essential autophagy proteins FIP200 or ATG9A exhibit overactivation and mislocalization of TBK1. By using both genetic and pharmacological inhibition of autophagy processes, we found that impaired autophagy leads to a significantly higher number of micronuclei – a hallmark for tumorigenesis that correlates with defects in mitosis and cytokinesis. Taken together our work has uncovered a novel function for the NAP1-TBK1 complex during mitosis and establishes that overactivation and mislocalization of TBK1 is a direct consequence of impaired autophagy which causes micronuclei formation.
- Remdesivir increases mtDNA copy number causing mild alterations to oxidative phosphorylationDeFoor, Nicole; Paul, Swagatika; Li, Shuang; Basso, Erwin K. Gudenschwager; Stevenson, Valentina; Browning, Jack L.; Prater, Anna K.; Brindley, Samantha; Tao, Ge; Pickrell, Alicia M. (Springer, 2023-12-01)SARS-CoV-2 causes the severe respiratory disease COVID-19. Remdesivir (RDV) was the first fast-tracked FDA approved treatment drug for COVID-19. RDV acts as an antiviral ribonucleoside (adenosine) analogue that becomes active once it accumulates intracellularly. It then diffuses into the host cell and terminates viral RNA transcription. Previous studies have shown that certain nucleoside analogues unintentionally inhibit mitochondrial RNA or DNA polymerases or cause mutational changes to mitochondrial DNA (mtDNA). These past findings on the mitochondrial toxicity of ribonucleoside analogues motivated us to investigate what effects RDV may have on mitochondrial function. Using in vitro and in vivo rodent models treated with RDV, we observed increases in mtDNA copy number in Mv1Lu cells (35.26% increase ± 11.33%) and liver (100.27% increase ± 32.73%) upon treatment. However, these increases only resulted in mild changes to mitochondrial function. Surprisingly, skeletal muscle and heart were extremely resistant to RDV treatment, tissues that have preferentially been affected by other nucleoside analogues. Although our data suggest that RDV does not greatly impact mitochondrial function, these data are insightful for the treatment of RDV for individuals with mitochondrial disease.
- Type I Interferon Response Is Mediated by NLRX1-cGAS-STING Signaling in Brain InjuryFritsch, Lauren E.; Ju, Jing; Basso, Erwin Kristobal Gudenschwager; Soliman, Eman; Paul, Swagatika; Chen, Jiang; Kaloss, Alexandra M.; Kowalski, Elizabeth A.; Tuhy, Taylor C.; Somaiya, Rachana Deven; Wang, Xia; Allen, Irving C.; Theus, Michelle H.; Pickrell, Alicia M. (Frontiers, 2022-02-25)Background: Inflammation is a significant contributor to neuronal death and dysfunction following traumatic brain injury (TBI). Recent evidence suggests that interferons may be a key regulator of this response. Our studies evaluated the role of the Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) signaling pathway in a murine model of TBI. Methods: Male, 8-week old wildtype, STING knockout (−/−), cGAS−/−, and NLRX1−/− mice were subjected to controlled cortical impact (CCI) or sham injury. Histopathological evaluation of tissue damage was assessed using non-biased stereology, which was complemented by analysis at the mRNA and protein level using qPCR and western blot analysis, respectively. Results: We found that STING and Type I interferon-stimulated genes were upregulated after CCI injury in a bi-phasic manner and that loss of cGAS or STING conferred neuroprotection concomitant with a blunted inflammatory response at 24 h post-injury. cGAS−/− animals showed reduced motor deficits 4 days after injury (dpi), and amelioration of tissue damage was seen in both groups of mice up to 14 dpi. Given that cGAS requires a cytosolic damage- or pathogen-associated molecular pattern (DAMP/PAMP) to prompt downstream STING signaling, we further demonstrate that mitochondrial DNA is present in the cytosol after TBI as one possible trigger for this pathway. Recent reports suggest that the immune modulator NLR containing X1 (NLRX1) may sequester STING during viral infection. Our findings show that NLRX1 may be an additional regulator that functions upstream to regulate the cGAS-STING pathway in the brain. Conclusions: These findings suggest that the canonical cGAS-STING-mediated Type I interferon signaling axis is a critical component of neural tissue damage following TBI and that mtDNA may be a possible trigger in this response.