Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion.

Ub to no good: How cytomegaloviruses exploit the ubiquitin proteasome system.

Human cytomegalovirus (HCMV) is a ubiquitous member of the Betaherpesvirinae subfamily, causing life-threatening diseases in individuals with impaired, immature, or senescent immunity. Accordingly, HIV-infected AIDS patients, transplant recipients, and congenitally infected neonates frequently suffer from symptomatic episodes of HCMV replication. Like all viruses, HCMV has a split relationship with the host proteome. Efficient virus replication can only be achieved if proteins involved in intrinsic, innate, and adaptive immune responses are sufficiently antagonized. Simultaneously, the abundance and function of proteins involved in the synthesis of chemical building blocks required for virus production, such as nucleotides, amino acids, and fatty acids, must be preserved or even enriched. The ubiquitin (Ub) proteasome system (UPS) constitutes one of the most relevant protein decay systems of eukaryotic cells. In addition to the regulation of the turn-over and abundance of thousands of proteins, the UPS also generates the majority of peptides presented by major histocompatibility complex (MHC) molecules to allow surveillance by T lymphocytes. Cytomegaloviruses exploit the UPS to regulate the abundance of viral proteins and to manipulate the host proteome in favour of viral replication and immune evasion. After summarizing the current knowledge of CMV-mediated misuse of the UPS, we discuss the evolution of viral proteins utilizing the UPS for the degradation of defined target proteins. We propose two alternative routes of adapter protein development and their mechanistic consequences.

Cell Death and Inflammation - A Vital but Dangerous Liaison.

The immune system has developed multiple ways to fight infection. Yet, it is constantly tasked with overcoming newly developing pathogenic mechanisms of resistance to host immunity. In most mammals, the stimulation of both innate and adaptive immune receptors can result in gene activation and cell death induction by apoptosis and necroptosis. RIPK1 and RIPK3 are key mediators of necroptosis; however, new findings support their role in the regulation of cell death-independent proinflammatory signaling. We discuss here the biological functions of RIPK1 and RIPK3, how they regulate cell death and inflammation, and the interplay between them. Finally, we discuss recent advances in our knowledge of linear ubiquitination which, alongside RIPK3 and caspase-8, exerts regulatory functions on RIPK1-mediated inflammation. Together, this review examines the complex interplay between RIPK1, RIPK3, and LUBAC that is important in regulating cell death and inflammatory signaling.

AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

As a master regulator for metabolic and energy homeostasis, AMPK controls the activity of metabolic enzymes and transcription factors in response to cellular ATP status. AMPK has been thus recognized as a main target for the regulation of cellular energy metabolism. Here, we report that AMPK can be down-regulated by the cullin-RING ubiquitin E3 ligase 4A (CRL4A) with cereblon (CRBN). CRL4A interacted with AMPK holoenzymes and mediated AMPKα-specific polyubiquitination for its proteasomal degradation through non-K48 polyubiquitin linkages. In the ubiquitination system, CRBN was required for efficient polyubiquitination of AMPKα subunits. Consistently, polyubiquitination of AMPKα subunits was reduced by inhibitors of CRL4A-CRBN. Physiologic function of AMPK down-regulation by CRL4-CRBN was also confirmed using mouse bone marrow-derived mast cells (BMMCs). The inactivation of CRL4A-CRBN in BMMC increased AMPK stability and suppressed secretion of allergic mediators via AMPK activation followed by MAPK inhibition. In addition, CRBN knockout of BMMC also decreased allergic responses in mice. Our results suggest that the CRL4A-CRBN axis could be a target for the regulation of AMPK-dependent responses.-Kwon, E., Li, X., Deng, Y., Chang, H. W., Kim, D. Y. AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins.

Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein.

Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes.

Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 µM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state.

Perturbation of cell cycle regulation triggers plant immune response via activation of disease resistance genes.

The Arabidopsis gene OSD1 (Omission of the Second Division) and its homolog UVI4 (UV-B-Insensitive 4) are negative regulators of anaphase-promoting complex/cyclosome (APC/C), a multisubunit ubiquitin E3 ligase that regulates the progression of cell cycles. Here we report the isolation of an activation tagging allele of OSD1 as an enhancer of a mutant of BON1 (BONZAI1), a negative regulator of plant immunity. Overexpression of OSD1 and UVI4 each leads to enhanced immunity to a bacterial pathogen, which is associated with increased expression of disease resistance (R) genes similar to the animal NOD1 receptor-like immune receptor genes. In addition, the reduction of function of one subunit of the APC complex APC10 exhibited a similar phenotype to that of overexpression of OSD1 or UVI4, indicating that altered APC function induces immune responses. Enhanced immune response induced by OSD1 overexpression is dependent on CYCB1;1, which is a degradation target of APC/C. It is also associated with up-regulation of R genes and is dependent on the R gene SNC1 (Suppressor of npr1-1, constitutive 1). Taken together, our findings reveal an unexpected link between cell cycle progression and plant immunity, suggesting that cell cycle misregulation could have an impact on expression of genes, including R genes, in plant immunity.

The E3 ubiquitin ligase Itch in T cell activation, differentiation, and tolerance.

Tagging a small molecule ubiquitin to a protein substrate, or protein ubiquitination, plays an important role in the immune responses. This process is catalyzed by a cascade of enzymatic reactions, with the E3 ubiquitin ligases being the critical enzymes that determine the specificity of substrate recognition. The E3 ligase Itch was identified from a mutant mouse which displays skin scratching and abnormal immune disorders. In the past few years, much progress has been made in our understanding of Itch-promoted protein ubiquitination, modulation of its ligase activity by upstream kinases, and the kinase-ligase interaction in T cell differentiation and tolerance induction.

Immunity by ubiquitylation: a reversible process of modification.

The conjugation of ubiquitin, a 76-amino-acid peptide, to a protein substrate provides a tag that either marks the labelled protein for degradation or modulates its function. The process of protein ubiquitylation--which is catalysed by coordinated enzymatic reactions that are mediated by enzymes known as E1, E2 and E3--has an important role in the modulation of immune responses. Importantly, protein ubiquitylation is a reversible process, and removal of ubiquitin molecules is mediated by de-ubiquitylating enzymes: for example, A20, which has been implicated in the regulation of immune responses. In addition, the conjugation of ubiquitin-like molecules, such as ISG15 (interferon-stimulated protein of 15 kDa), to proteins is also involved in immune regulation. This Review covers recent progress in our understanding of protein ubiquitylation in the immune system.

Large-scale purification of the vertebrate anaphase-promoting complex/cyclosome.

The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle. The APC/C is a 1.5-MDa complex composed of at least 12 different core subunits. At different stages of mitosis and G1, the APC/C associates with a variety of regulatory proteins, such as the activator proteins Cdc20 and Cdh1 and the mitotic checkpoint complex (MCC), which regulate APC/C activity in a substrate-specific manner. Although APC/C and its regulators have been under intense investigation, it is still poorly understood how substrates are recognized and ubiquitinated by the APC/C, why so many subunits are required for these processes, and how regulators of the APC/C control its ubiquitin ligase activity in a substrate-specific manner. This chapter describes a simple and rapid procedure that allows the isolation of APC/C from vertebrate cells and tissues with reasonable purity and at high concentrations, yielding up to 0.5 mg of APC/C. This procedure should facilitate biochemical, biophysical, and structural analyses of the APC/C that will be needed for a better mechanistic understanding of its function and regulation.

Immunologic aspects of protein degradation by the ubiquitin-proteasome system.

The ubiquitin-proteasome pathway has a central role in selective degradation of intracellular proteins. Among the key proteins degraded by the system are those involved in the control of inflammation, cell cycle regulation and gene expression. With numerous important cellular pathways affected, derangements in the ubiquitin system were shown to result in a variety of human diseases including malignancies, neurodegenerative diseases and hereditary syndromes, and proteasome inhibition was implicated as a potential treatment for cancer and inflammatory conditions. Two proteasome inhibitors are currently under clinical evaluation for multiple myeloma and acute ischemic stroke. The ubiquitin system also has an important function in the immune and inflammatory response. It is involved in antigen processing and presentation to cytotoxic T cells, and the activation of nuclear factor-kappa B--the central transcription factor of the immune system. Since the proteasome is the central source of antigenic peptides that are presented to the immune system, some viruses, such as the Epstein-Barr virus, developed escape mechanisms that manipulate the ubiquitin-proteasome system in order to persist in the infected host. Understanding the mechanisms underlying the production of viral antigens by the ubiquitin-proteasome system may have therapeutic applications such as future development of vaccines.

Ubiquitin ligases and the immune response.

Ubiquitin (Ub)-protein conjugation represents a novel means of posttranscriptional modification in a proteolysis-dependent or -independent manner. E3 Ub ligases play a key role in governing the cascade of Ub transfer reactions by recognizing and catalyzing Ub conjugation to specific protein substrates. The E3s, which can be generally classified into HECT-type and RING-type families, are involved in the regulation of many aspects of the immune system, including the development, activation, and differentiation of lymphocytes, T cell-tolerance induction, antigen presentation, immune evasion, and virus budding. E3-promoted ubiquitination affects a wide array of biological processes, such as receptor downmodulation, signal transduction, protein processing or translocation, protein-protein interaction, and gene transcription, in addition to proteasome-mediated degradation. Deficiency or mutation of some of the E3s like Cbl, Cbl-b, or Itch, causes abnormal immune responses such as autoimmunity, malignancy, and inflammation. This review discusses our current understanding of E3 Ub ligases in both innate and adaptive immunity. Such knowledge may facilitate the development of novel therapeutic approaches for immunological diseases.