Biology subjects

Pfeffer, L. M.

Publications and source records attributed to Pfeffer, L. M..

3 recordsLinked to original sources

bioRxiv2023

Ccr4-Not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy

The Ccr4-Not complex containing the Not4 ubiquitin ligase regulates gene transcription and mRNA decay, yet it also has poorly defined roles in translation, proteostasis, and endolysosomal-dependent nutrient signaling. To define how Ccr4-Not mediated ubiquitin signaling regulates these additional processes, we performed quantitative proteomics in the yeast Saccharomyces cerevisiae lacking the Not4 ubiquitin ligase, and also in cells overexpressing either wild-type or functionally inactive ligase. Herein, we provide evidence that both increased and decreased Ccr4-Not ubiquitin signaling disrupts ribosomal protein (RP) homeostasis independently of reduced RP mRNA changes or reductions in known Not4 ribosomal substrates. Surprisingly, we also find that both Not4-mediated ubiquitin signaling, and the Ccr4 subunit, actively inhibit 40S ribosomal autophagy. This 40S autophagy is independent of canonical Atg7-dependent macroautophagy, thus indicating microautophagy activation is responsible. Furthermore, the Not4 ligase genetically interacts with endolysosomal pathway effectors to control both RP expression and 40S autophagy efficiency. Overall, we demonstrate that balanced Ccr4-Not ligase activity maintains RP homeostasis, and that Ccr4-Not ubiquitin signaling interacts with the endolysosomal pathway to both regulate RP expression and inhibit 40S ribosomal autophagy.

molecular biology↗

bioRxiv2022

Combination of Volasertib and Rapamycin Inhibits the Regrowth of TSC2-Deficient Tumors

Mutations in TSC1 and TSC2 lead to hyperactivation of mTORC1 and cause Tuberous Sclerosis Complex (TSC) and pulmonary Lymphangioleiomyomatosis (LAM). Rapamycin and rapalogs are potent inhibitors of mTORC1 activity and are approved for the treatment of TSC and LAM. Nevertheless, rapalogs do not cause tumor cell death, and cessation of therapy leads to tumor regrowth. Polo-like kinase 1 (PLK1) interacts with and phosphorylates TSC1, and PLK1 inhibition induces apoptosis and attenuates autophagy in TSC1/TSC2-deficient cells. Here we report that the PLK1 inhibitor volasertib decreases the viability and survival and induces apoptosis in 621-101 cells, a TSC-deficient renal angiomyolipoma cell line from a LAM patient. Combined with rapamycin, volasertib further decreased the survival of 621-101 cells. In vivo, volasertib reduced short-term mouse lung colonization by TSC2-deficient cells and decreased the growth of TSC2-deficient subcutaneous tumors. Mice treated with a combination of volasertib and rapamycin had slower tumor relapse after discontinuation of treatment, compared to rapamycin only. Approximately 35 days after discontinuation of treatment, we observed persistent apoptotic markers and gene expression changes for type I interferon signaling in the regrowth tumors from combination-treated mice, compared to rapamycin only. Notably, combination treatment potently inhibited the growth of subcutaneous tumors derived from the rapamycin-refractory cell line ELT3-245. Taken together, our current work demonstrates that for the management of TSC and LAM disease combination of mTORC1 and PLK1 inhibitors in some cases may be advantageous over currently used rapalog monotherapies.

cancer biology↗

bioRxiv2022

STAT3 regulates cytokine production downstream of TNFR1 in part by inducing expression of TNFAIP3/A20

BackgroundPrevious work demonstrated that the Signal Transducer and Activator of Transcription 3 (STAT3) is activated downstream of the Type 1 TNF Receptor. However, whether and how STAT3 regulates gene expression downstream of TNFR1 has not been elucidated. MethodsGlobal transcriptome analysis by RNA sequencing was performed in wild type and STAT3 knockout mouse embryonic fibroblasts (MEFs) stimulated with TNF. The fold changes in gene expression were assessed bioinformatically. Results of the RNA sequencing were validated at the protein level by using multiplex cytokine assays and immunoblotting. ResultsStimulation of MEFs with TNF or an agonist antibody to TNFR1 activated STAT3, and this was inhibited by pharmacological inhibition of Jak2 and cSrc. At 4 hours after TNF stimulation, STAT3 knockout MEFs had a greater level than WT MEFs of induction of the chemokines Ccl2, Cxcl1 and Cxcl10 at the RNA and protein levels. Mechanistically, this was due STAT3 promoting the expression of Tnfaip3/A20, a ubiquitin modifying enzyme that inhibits inflammation, in wild-type MEFs at early timepoints after TNFR1 stimulation. In STAT3 knockout MEFs TNF failed to induce the expression of Tnfaip3/A20 or GM-CSF when acting through TNFR1. Expression of A20 into STAT3 knockout MEFs suppressed cytokine expression. ConclusionSTAT3 limits the induction of Ccl2, Cxcl1 and Cxcl10 in response to TNFR1 activation by promoting the expression of Tnfaip3/A20. On the other hand, STAT3 promotes the expression of GM-CSF in response to TNFR1 stimulation. These results show that STAT3 modulates inflammatory signaling by TNF in normal cells.

cell biology↗