Disordered regions in the IRE1α ER lumenal domain mediate its stress-induced clustering.

Conserved signaling cascades monitor protein-folding homeostasis to ensure proper cellular function. One of the evolutionary conserved key players is IRE1, which maintains endoplasmic reticulum (ER) homeostasis through the unfolded protein response (UPR). Upon accumulation of misfolded proteins in the ER, IRE1 forms clusters on the ER membrane to initiate UPR signaling.

SPOP targets the immune transcription factor IRF1 for proteasomal degradation.

Adaptation of the functional proteome is essential to counter pathogens during infection, yet precisely timed degradation of these response proteins after pathogen clearance is likewise key to preventing autoimmunity. Interferon regulatory factor 1 (IRF1) plays an essential role as a transcription factor in driving the expression of immune response genes during infection. The striking difference in functional output with other IRFs is that IRF1 also drives the expression of various cell cycle inhibiting factors, making it an important tumor suppressor.

HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation.

Tristetraprolin (TTP) is a critical negative immune regulator. It binds AU-rich elements in the untranslated-regions of many mRNAs encoding pro-inflammatory mediators, thereby accelerating their decay. A key but poorly understood mechanism of TTP regulation is its timely proteolytic removal: TTP is degraded by the proteasome through yet unidentified phosphorylation-controlled drivers.

Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins.

AKIRIN2 controls the nuclear import of proteasomes in vertebrates.

Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth.

Lipocalin 2 modulates dendritic cell activity and shapes immunity to influenza in a microbiome dependent manner.

Lipocalin 2 (LCN2) is a secreted glycoprotein with roles in multiple biological processes. It contributes to host defense by interference with bacterial iron uptake and exerts immunomodulatory functions in various diseases. Here, we aimed to characterize the function of LCN2 in lung macrophages and dendritic cells (DCs) using Lcn2-/- mice.

Negative Regulation of the Innate Immune Response through Proteasomal Degradation and Deubiquitination.

The rapid and dynamic activation of the innate immune system is achieved through complex signaling networks regulated by post-translational modifications modulating the subcellular localization, activity, and abundance of signaling molecules. Many constitutively expressed signaling molecules are present in the cell in inactive forms, and become functionally activated once they are modified with ubiquitin, and, in turn, inactivated by removal of the same post-translational mark. Moreover, upon infection resolution a rapid remodeling of the proteome needs to occur, ensuring the removal of induced response proteins to prevent hyperactivation.

A repetitive acidic region contributes to the extremely rapid degradation of the cell-context essential protein TRIM52.

Tripartite motif protein 52 (TRIM52) is a non-canonical TRIM family member harbouring the largest RING domain encoded in the human genome. In humans TRIM52 is conserved and has been under positive selection pressure, yet it has been lost in many non-primates. Competitive cell fitness assays demonstrated that TRIM52 ablation reduces cellular fitness in multiple different cell types.

TRIM proteins.

Vunjak and Versteeg introduce the TRIM family of post-translational modifiers and the roles of these proteins in viral restriction, immune signaling and autophagy.

Human tripartite motif protein 52 is required for cell context-dependent proliferation.

Tripartite motif (TRIM) proteins have been shown to play important roles in cancer development and progression by modulating cell proliferation or resistance from cell death during non-homeostatic stress conditions found in tumor micro-environments. In this study, we set out to investigate the importance for cellular fitness of the virtually uncharacterized family member TRIM52. The human gene has arisen recently in evolution, making it unlikely that TRIM52 is required for basic cellular functions in normal cells.

Ubiquitin enzymes in the regulation of immune responses.

Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed.

Negative regulation of NF-κB activity by brain-specific TRIpartite Motif protein 9.

The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The β-transducin repeat-containing protein (β-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. β-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation.

InTRIMsic immunity: Positive and negative regulation of immune signaling by tripartite motif proteins.

During the immune response, striking the right balance between positive and negative regulation is critical to effectively mount an anti-microbial defense while preventing detrimental effects from exacerbated immune activation. Intra-cellular immune signaling is tightly regulated by various post-translational modifications, which allow for this dynamic response. One of the post-translational modifiers critical for immune control is ubiquitin, which can be covalently conjugated to lysines in target molecules, thereby altering their functional properties.

Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase-mediated antiviral response.

Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε).

TRIMmunity: the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity.

Tripartite motif (TRIM) proteins have been implicated in multiple cellular functions, including antiviral activity. Research efforts so far indicate that the antiviral activity of TRIMs relies, for the most part, on their function as E3-ubiquitin ligases. A substantial number of the TRIM family members have been demonstrated to mediate innate immune cell signal transduction and subsequent cytokine induction.

The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors.

Innate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs.

Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling.

Regulation of the innate immune system by ubiquitin and ubiquitin-like modifiers.

Detection of invading pathogens by pattern recognition receptors (PRRs) is crucial for the activation of the innate immune response. These sensors signal through intertwining signaling cascades which result in the expression of pro-inflammatory cytokines and type I interferons. Conjugation, or binding, of ubiquitin and ubiquitin-like modifiers (UBLs) to a plethora of immune signaling molecules forms a common theme in innate immune regulation.

HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells.

Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not.

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor.

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV).

Viral tricks to grid-lock the type I interferon system.

Type I interferons (IFNs) play a crucial role in the innate immune avant-garde against viral infections. Virtually all viruses have developed means to counteract the induction, signaling, or antiviral actions of the IFN circuit. Over 170 different virus-encoded IFN antagonists from 93 distinct viruses have been described up to now, indicating that most viruses interfere with multiple stages of the IFN response.

Species-specific antagonism of host ISGylation by the influenza B virus NS1 protein.

Interferon-stimulated expression and conjugation of the ubiquitin-like modifier ISG15 restricts replication of several viruses. Here, we established complete E1-activating, E2-conjugating, and E3 ligase-dependent expression systems for assaying both human and mouse ISGylation. We confirm that human HerC5, but not human HerC6, has ISG15 E3 ligase activity and identify mouse HerC6 as a bona fide ISG15 E3 ligase.

The coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations.

Murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS) coronavirus (CoV) are two of the best-studied representatives of the family Coronaviridae. During CoV infection, numerous cytokines and chemokines are induced in vitro and in vivo. Human interleukin 8 and its mouse functional counterpart, CXCL2, are early-expressed chemokines.

Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition.

Many viruses encode antagonists to prevent interferon (IFN) induction. Infection of fibroblasts with the murine hepatitis coronavirus (MHV) and SARS-coronavirus (SARS-CoV) did not result in nuclear translocation of interferon-regulatory factor 3 (IRF3), a key transcription factor involved in IFN induction, and induction of IFN mRNA transcription. Furthermore, MHV and SARS-CoV infection could not prevent IFN induction by poly (I:C) or Sendai virus, suggesting that these CoVs do not inactivate IRF3-mediated transcription regulation, but apparently prevent detection of replicative RNA by cellular sensory molecules.

Transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells.

Understanding the orchestrated genome-wide cellular responses is critical for comprehending the early events of coronavirus infection. Microarray analysis was applied to assess changes in cellular expression profiles during different stages of two independent, highly controlled murine hepatitis virus (MHV) infections in vitro. Fibroblast-like L cells were infected at high multiplicity in order to study the direct effects of a synchronized lytic coronavirus infection.