Autophagy and interferon (IFN)-mediated innate immunity are critical antiviral defense mechanisms, and recent evidence indicated that tripartite motif (TRIM) proteins are important regulators of both processes. While the role of TRIM proteins in modulating antiviral cytokine responses has been well established, much less is known about their involvement in autophagy in response to different viral pathogens. Through a targeted RNAi screen examining the relevance of selected TRIM proteins in autophagy induced by herpes simplex virus 1 (HSV-1), encephalomyocarditis virus (EMCV), and influenza A virus (IAV), we identified several TRIM proteins that regulated autophagy in a virus-species specific manner, as well as a few TRIM proteins that were essential for autophagy triggered by all three viruses and rapamycin, among them TRIM23. TRIM23 was critical for autophagy-mediated restriction of multiple viruses, and this activity was dependent on both its RING E3 ligase and ADP-ribosylation factor (ARF) GTPase activity. Mechanistic studies revealed that unconventional K27-linked auto-ubiquitination of the ARF domain is essential for the GTP hydrolysis activity of TRIM23 and activation of TANK-binding kinase 1 (TBK1) by facilitating its dimerization and ability to phosphorylate the selective autophagy receptor p62. Our work identifies the TRIM23-TBK1-p62 axis as a key component of selective autophagy and further reveals a role for K27-linked ubiquitination in GTPase-dependent TBK1 activation.

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys63)–linked ubiquitin chains to the RNA sensor retinoic acid–inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys48-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys48-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I–dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I–mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling.

The cytosolic sensor MDA5 is crucial for antiviral innate immune defense against various RNA viruses including measles virus; as such, many viruses have evolved strategies to antagonize the antiviral activity of MDA5. Here, we show that measles virus escapes MDA5 detection by targeting the phosphatases PP1α and PP1γ, which regulate MDA5 activity by removing an inhibitory phosphorylation mark. The V proteins of measles virus and the related paramyxovirus Nipah virus interact with PP1α/γ, preventing PP1-mediated dephosphorylation of MDA5 and thereby its activation. The PP1 interaction with the measles V protein is mediated by a conserved PP1-binding motif in the C-terminal region of the V protein. A recombinant measles virus expressing a mutant V protein deficient in PP1 binding is unable to antagonize MDA5 and is growth-impaired due to its inability to suppress interferon induction. This identifies PP1 antagonism as a mechanism employed by paramyxoviruses for evading innate immune recognition.

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Author contributions S.N. and W.G. conceived and designed the overall project. S.S. and C.I.U. assisted with selecting the family, gathering the clinical histories and collecting DNA samples under human subject IRB-approved protocols. S.N., W.G. and I.L. designed the WGS analysis. I.L. performed the WGS analysis and candidate variant filtering. S.N., J.W., A.J.K., J.E.H., A.G.C. and J.H. designed and generated the zebrafish rabl3 mutant lines and performed the cancer studies. J.R.H. and S.N. performed zebrafish histology preparation and analysis. J.D.M. performed and analyzed the AP-MS experiments and CompPASS suite protein interactomics. S.N., W.G. and C.W. conceived and designed the in vitro immunoprecipitation, prenylation assays and HEK293T cell proliferation assays, and P.G., A.B., E.L. and B.U. performed these experiments. S.N. and O.M. designed and performed RASless MEF experiments. J.W.P. performed protein structural modeling. B.C.J. and C.A.F. designed and performed purification of recombinant protein. J.A.P., S.G. and J.D.M. assisted with mass spectrometry analysis. Y.H. assisted with RNA-seq data analysis. M.B.G. performed the zebrafish μCT and bone histomorphometric analysis. O.M., X.W. and J.D.M. provided assistance with tissue culture experiments. C.A.C. and J.A.R. provided analysis of clinical exome sequencing data. C.A.C. and I.L. provided analysis of variants in the Exome Aggregation Consortium.

Ncoa4 mediates autophagic degradation of ferritin, the cytosolic iron storage complex, to maintain intracellular iron homeostasis. Recent evidence also supports a role for Ncoa4 in systemic iron homeostasis and erythropoiesis. However, the specific contribution and temporal importance of Ncoa4-mediated ferritinophagy in regulating systemic iron homeostasis and erythropoiesis is unclear. Here, we show that Ncoa4 has a critical role in basal systemic iron homeostasis and both cell autonomous and non-autonomous roles in murine erythropoiesis. Using an inducible murine model of Ncoa4 knockout, acute systemic disruption of Ncoa4 impaired systemic iron homeostasis leading to tissue ferritin and iron accumulation, a decrease in serum iron, and anemia. Mice acutely depleted of Ncoa4 engaged the Hif2a-erythropoietin system to compensate for anemia. Mice with targeted deletion of Ncoa4 specifically in the erythroid compartment developed a pronounced anemia in the immediate postnatal stage, a mild hypochromic microcytic anemia at adult stages, and were more sensitive to hemolysis with higher requirements for the Hif2a-erythropoietin axis and extramedullary erythropoiesis during recovery. These studies demonstrate the importance of Ncoa4-mediated ferritinophagy as a regulator of systemic iron homeostasis and define the relative cell autonomous and non-autonomous contributions of Ncoa4 in supporting erythropoiesis in vivo .

Covalent inhibitors of the KRAS G12C oncoprotein have recently been developed and are being evaluated in clinical trials. Resistance to targeted therapies is common and likely to limit long-term efficacy of KRAS inhibitors (KRASi). To identify pathways of adaptation to KRASi and to predict drug combinations that circumvent resistance, we used a mass spectrometry-based quantitative temporal proteomics and bioinformatics workflow to profile the temporal proteomic response to KRAS G12C inhibition in pancreatic and lung cancer 2D and 3D cellular models. We quantified 10,805 proteins across our datasets, representing the most comprehensive KRASi proteomics effort to date. Our data reveal common mechanisms of acute and long-term response between KRAS G12C -driven tumors. To facilitate discovery in the cancer biology community, we generated an interactive 'KRASi proteome' website (https://manciaslab.shinyapps.io/KRASi/). Based on these proteomic data, we identified potent combinations of KRASi with PI3K, HSP90, CDK4/6, and SHP2 inhibitors, in some instances converting a cytostatic response to KRASi monotherapy to a cytotoxic response to combination treatment. Overall, using our quantitative temporal proteomics-bioinformatics platform, we have comprehensively characterized the proteomic adaptations to KRASi and identified combinatorial regimens to induce cytotoxicity with potential therapeutic utility.

We thank Nai and colleagues for their interest in our recently published paper, Santana-Codina et al., 1 and their comment, also published in Haematologica. 2 Their comment, containing new unpublished experimental data, questions the importance of nuclear receptor coactivator 4 (NCOA4) in acute erythropoietic expansion. Here, we respond to clarify inconsistencies in the interpretation of their new data with respect to our experiments and highlight the multiple lines of evidence that support our overall conclusions regarding the cell autonomous and non-autonomous roles of NCOA4 in supporting murine erythropoiesis. With respect to their concerns over the use of tamoxifen in our inducible model of acute whole body Ncoa4 genetic ablation: we stand by the conclusions of our experiments as they were tightly controlled with all appropriate genetic backgrounds and treatment controls utilized. While we agree that tamoxifen may have off-target effects on red blood cells (RBC), our experiments as designed and carried out were well-controlled. Specifically, mice in each group had no baseline alterations in systemic iron parameters or erythropoiesis prior to tamoxifen administration making the alterations observed most likely to be a result of acute Ncoa4 ablation leading to an acute loss of flux of ferritin through the autophagic degradation pathway thereby altering systemic iron homeostasis and basal erythropoiesis. The key difference in the new data presented by Nai and colleagues in their comment 2 is the presence of long-standing Ncoa4 knockout in their mouse model prior to administration of tamoxifen, which we argue makes their conclusions uninterpretable with respect to our data in an inducible knockout mouse model. Specifically, they use a mouse model with constitutive (from birth) total body knockout of Ncoa4, which is a distinct situation compared to acute ablation of Ncoa4 expression in an adult mice. The critical point here is that with a constitutive total body ablation of Ncoa4 from birth, erythropoiesis is altered in such a way that RBC at the time of tamoxifen administration already have accumulated long-standing significant changes that alter the baseline response to any agent that has potential deleterious effects on the RBC, such as tamoxifen. For that matter, Nai et al. do not present hemoglobin levels in wild-type mice that were subjected to gavage solely with the vehicle (corn oil) that was used to dissolve tamoxifen. Given that repeated gavage is expected to induce a profound stress response, it is unclear if the reduction in hemoglobin levels observed in wild type mice (and which form the basis of their arguments) can be attributed specifically to tamoxifen. While we cannot fully evaluate their claim regarding the lack of alterations in systemic iron parameters in the Sv129/J background they use given their reference to unpublished data, we do note that these Ncoa4 knockout animals appear to have altered baseline hematologic parameters with microcytosis and a trend towards decreased hemogolobin and he...