Regulation of Mitochondria-Derived Immune Activation by 'Antiviral' TRIM Proteins.

Mitochondria are key orchestrators of antiviral responses that serve as platforms for the assembly and activation of innate immune-signaling complexes. In response to viral infection, mitochondria can be triggered to release immune-stimulatory molecules that can boost interferon production. These same molecules can be released by damaged mitochondria to induce pathogenic, antiviral-like immune responses in the absence of infection. This review explores how members of the tripartite motif-containing (TRIM) protein family, which are recognized for their roles in antiviral defense, regulate mitochondria-based innate immune activation. In antiviral defense, TRIMs are essential components of immune signal transduction pathways and function as directly acting viral restriction factors. TRIMs carry out conceptually similar activities when controlling immune activation related to mitochondria. First, they modulate immune-signaling pathways that can be activated by mitochondrial molecules. Second, they co-ordinate the direct removal of mitochondria and associated immune-activating factors through mitophagy. These insights broaden the scope of TRIM actions in innate immunity and may implicate TRIMs in diseases associated with mitochondria-derived inflammation.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α. Mitochondrial damage triggers TRIM5α's auto-ubiquitination and its interaction with ubiquitin-binding autophagy adaptors including NDP52, optineurin, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1. TRIM5α with intact ubiquitination function is required for the proper accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Additionally, we show that TRIM5α can directly recruit autophagy initiation machinery to damaged mitochondria. Our data support a model in which TRIM5α provides a self-amplifying, mitochondria-localized, ubiquitin-based, assembly platform for TBK1 and mitophagy adaptors that is ultimately required to recruit the core autophagy machinery.

Interactomic analysis reveals a homeostatic role for the HIV restriction factor TRIM5α in mitophagy.

The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.

Pathogen size alters C-type lectin receptor signaling in dendritic cells to influence CD4 Th9 cell differentiation.

Dectin-1 recognizes ß-glucan in fungal cell walls, and activation of Dectin-1 in dendritic cells (DCs) influences immune responses against fungi. Although many studies have shown that DCs activated via Dectin-1 induce different subsets of T helper cells according to different cytokine milieus, the mechanisms underlying such differences remain unknown. By harnessing polymorphic Candida albicans and polystyrene beads of different sizes, we find that target size influences production of cytokines that control differentiation of T helper cell subsets. Hyphal C. albicans and large beads activate DCs but cannot be phagocytosed due to their sizes, which prolongs the duration of Dectin-1 signaling. Transcriptomic analysis reveals that expression of Il33 is significantly increased by larger targets, and increased IL-33 expression promotes TH9 responses. Expression of IL-33 is regulated by the Dectin-1-SYK-PLCγ-CARD9-ERK pathway. Altogether, our study demonstrates that size of fungi can be a determining factor in how DCs induce context-appropriate adaptive immune responses.

Distinct and sequential re-replication barriers ensure precise genome duplication.

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.

Longitudinal trends of blood lead levels before and after leaded gasoline regulation in Korea.

The objective of this study was to verify a change in the longitudinal trend of blood lead levels for the Korean population, before and after the regulation of leaded gasoline- which occurred between 1987 and 1993 in Korea. A total of 77 reports on blood lead levels among general Korean population between 1981 and 2014 were selected, and the results were summarized to have the variables of year, number of subjects, the subjects' range in age, gender, and blood lead concentrations (arithmetic mean). The annual average atmospheric lead levels for four major cities (i.e., Seoul, Busan, Daegu and Gwangju) were collected from the Air Pollution Monitoring Database from 1991, and pilot studies from 1985 to 1990 before the national air quality monitoring system was launched in 1991. Blood lead levels were visualized in a bubble plot in which the size of each bubble represented the sample size of each study, and the annual average concentrations in ambient air were depicted on line graphs. Blood lead levels in the Korean population tended to gradually increase from the early 1980s (approximately 15-20 µg/dL) until 1990-1992 (20-25 µg/dL). Blood lead levels then began to rapidly decrease until 2014 (<2 µg/dL). Similar patterns were observed for both adults (≥20 years) and younger children/adolescents. The same longitudinal trend was observed in annual average atmospheric lead concentration, which suggests a significant correlation between air lead concentration and blood lead concentration in the general population. In conclusion, the regulation of leaded gasoline has significantly contributed to the rapid change in blood lead concentrations. And, the regulation of other sources of lead exposure should be considered to further decrease blood lead levels in the Korean population.

Host-microbe interactions: commensal fungi in the gut.

Fungi are ubiquitous microbes that are common in diverse environments including as commensal organisms on the human body. In addition to its obvious role as a digestive organ, the intestines have been further appreciated as important for the development, maintenance, and instruction of the immune system. The gut harbors many types of microorganisms including bacteria, archaea, fungi, and viruses, and many studies over the past couple of decades have documented an important role for intestinal bacteria in immunological function. Recent studies are now suggesting that intestinal fungi (the gut 'mycobiome') may similarly play important roles in host immunity and inflammation. This review will discuss recent studies that will influence our growing understanding of the role(s) of intestinal fungi in health and disease.

CDK1-dependent inhibition of the E3 ubiquitin ligase CRL4CDT2 ensures robust transition from S Phase to Mitosis.

Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4(CDT2) ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNA(DNA)) triggers the interaction between CRL4(CDT2) and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNA(DNA) is no longer sufficient to trigger CRL4(CDT2)-mediated degradation. A CDK1-dependent mechanism that blocks CRL4(CDT2) activity by interfering with CDT2 recruitment to chromatin actively protects CRL4(CDT2) substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4(CDT2) inactivation contributes to efficient transition from S phase to mitosis.

MEKK1/MEKK4 are responsible for TRAIL-induced JNK/p38 phosphorylation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to activate mitogen-activated protein kinases (MAPKs) depending on caspase and mammalian sterile 20-like kinase 1 activations. However, the upstream molecule of MAPKs has not yet been identified. The mitogen-activated protein kinase kinase 1 (MEKK1) and the apoptosis signal-regulating kinase 1 (ASK1) are considered to be possible candidates for the action of MAPKKKs induced by TRAIL and the possibility of reactive oxygen species involvement has also been investigated. We found that MEKK1/MEKK4 as opposed to ASK1, are responsible for TRAIL-induced c-Jun NH2-terminal kinase (JNK) or p38 activation, and that their catalytic activity is repressed by the caspase-8 inhibitor, suggesting that the caspase-8 activation induced by TRAIL is indispensible for MEKK activation. The 14-3-3 θ was also shown to interact with and to dissociate from MEKK1 by TRAIL treatment, thus implicating the 14-3-3 protein as a negative regulator of MEKK1 activation. Taken together, we show herein that the upstream molecule of the TRAIL-induced MAPK activation is MEKK, as opposed to ASK1, via the mediation of its signal through JNK/p38 in a caspase-8-dependent manner.

TRAIL-induced caspase/p38 activation is responsible for the increased catalytic and invasive activities of Akt.

We previously observed that TRAIL induces acquired TRAIL resistance coinciding with increased Akt phosphorylation brought about by the Src-PI3K-Akt signaling pathways and mediated by c-Cbl. c-Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is simultaneously involved in the rapid degradation of TRAIL receptors and Akt phosphorylation during TRAIL treatment. Here, we show that Akt phosphorylation is not exclusively responsible for acquired TRAIL resistance. Akt catalytic activation is known to increase during metabolic oxidative stress, but we show that TRAIL also dramatically induces the catalytic activation of Akt in TRAIL-sensitive cells, but not in TRAIL-resistant cells. This suggests that Akt catalytic activation during TRAIL-induced apoptosis is likely to play a compensatory role in the maintenance of cell homeostasis. In addition, activated p38 and phosphorylated HSP27 were found to act as downstream effector molecules of p38 during TRAIL treatment and were shown to be responsible for increased Akt catalytic and invasive activities.