Krüger, E. | bioRxiv Science

bioRxiv2022

Proteasome dysfunction disrupts adipogenesis and induces inflammation via ATF3

ObjectiveRegulation of proteasomal activity is an essential component of cellular proteostasis and function. This is evident in patients with mutations in proteasome subunits and regulators, who suffer from proteasome-associated autoinflammatory syndromes (PRAAS). These patients display lipodystrophy and fevers, which may be partly related to adipocyte malfunction and abnormal thermogenesis in adipose tissue. However, the cell-intrinsic pathways that could underlie these symptoms are unclear. Here, we investigate the impact of two proteasome subunits implicated in PRAAS, Psmb4 and Psmb8, on differentiation, function and proteostasis of brown adipocytes. MethodsIn immortalized mouse brown pre-adipocytes, levels of Psmb4, Psmb8, and downstream effectors genes were downregulated through reverse transfection with siRNA. Adipocytes were differentiated and analyzed with various assays of adipogenesis, lipogenesis, lipolysis, inflammation, and respiration. ResultsLoss of Psmb4, but not Psmb8, disrupted proteostasis and adipogenesis. Proteasome function was reduced upon Psmb4 loss, but partly recovered by the activation of Nuclear factor, erythroid-2, like-1 (Nfe2l1). In addition, cells displayed higher levels of surrogate inflammation and stress markers, including Activating transcription factor-3 (Atf3). Simultaneous silencing of Psmb4 and Atf3 lowered inflammation and restored adipogenesis. ConclusionsOur study shows that Psmb4 is required for adipocyte development and function in cultured adipocytes. These results imply that in humans with PSMB4 mutations, PRAAS-associated lipodystrophy is partly caused by disturbed adipogenesis. While we uncover a role for Nfe2l1 in the maintenance of proteostasis under these conditions, Atf3 is a key effector of inflammation and blocking adipogenesis. In conclusion, our work highlights how proteasome dysfunction is sensed and mitigated by the integrated stress response in adipocytes with potential relevance for PRAAS patients and beyond. Highlights- PRAAS-associated PSMB4 is required for brown adipocyte differentiation - Loss of PSMB4 activates NFE2L1 to counteract proteasome dysfunction - The ATF3 pathway regulates adipocyte dysfunction and inflammation - Loss of ATF3 restores adipogenesis in cells with loss of PSMB4 Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/476367v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@4c9c5eorg.highwire.dtl.DTLVardef@1ae0674org.highwire.dtl.DTLVardef@29c741org.highwire.dtl.DTLVardef@d8a584_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2020

DDI2 protease activity controls embryonic development and inflammation via TCF11/NRF1

DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the ER-bound transcription factor TCF11/NRF1 (NFE2L1), a master regulator of proteostasis maintenance in mammalian cells, and ensures the expression of rescue factors including proteasome subunits. Here we describe the consequences of DDI2 ablation both in vivo and in cells. Knock-out of DDI2 in mice resulted in embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of the embryos and surrogate DDI2 knock-out cell lines showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates, and induction of the unfolded protein and integrated stress responses. We also show that DDI2 KO-induced proteotoxic stress causes the cell-autonomous innate immune system to induce a type I interferon signature. These results indicate an important role for DDI2 in the proteostasis network of cells and tissues and in the maintenance of a balanced immune response. HighlightsO_LIDDI2-deficiency in mice causes severe developmental failure and embryonic lethality at mid-late gestation C_LIO_LIDDI2-deficiency causes severe proteotoxic stress and proteasome impairment C_LIO_LIDDI2-deficiency induces the UPR and ISR signaling pathways C_LIO_LIDDI2-deficient cells survive via STAT3-dependent interferon signaling C_LI

cell biology↗