Mitophagy facilitates the selective elimination of impaired or depolarized mitochondria through targeting the latter to autophagosomes. Parkin becomes localized to depolarized mitochondria in ...a PINK1-dependent manner and polyubiquitinates multiple mitochondrial outer membrane proteins. This permits ubiquitin-binding proteins (e.g., p62 and NBR1) to target impaired mitochondria to autophagosomes via Atg8/LC3II. Bcl-2 family proteins regulate mitochondrial outer membrane permeabilization during apoptosis and can also influence macroautophagy via interactions with Beclin-1. Here, we show that Parkin-dependent mitophagy is antagonized by prosurvival members of the Bcl-2 family (e.g., Bcl-xL and Mcl-1) in a Beclin-1-independent manner. Bcl-2 proteins suppressed mitophagy through inhibition of Parkin translocation to depolarized mitochondria. Consistent with this, Parkin translocation to mitochondria was enhanced by BH3-only proteins or a BH3-only mimetic. Taken together with their role as regulators of apoptosis-associated mitochondrial permeabilization, as well as mitochondrial fission/fusion dynamics, this suggests that Bcl-2 family proteins act as global regulators of mitochondrial homeostasis.
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•Bcl-2 family proteins regulate mitochondrial quality control•Prosurvival Bcl-2 family proteins antagonize Parkin-dependent mitophagy•Bcl-2 family proteins influence Parkin translocation to depolarized mitochondria•Prosurvival Bcl-2 family proteins interact with Parkin
Parkin, the ubiquitin ligase that is mutated in Parkinson’s disease, regulates the clearance of damaged mitochondria through selective autophagy (mitophagy). Here, Hollville et al. show that Parkin-dependent mitophagy is regulated by members of the Bcl-2 family, which are more commonly known for their ability to regulate apoptotic cell death.
Mitochondrial depolarization promotes Parkin- and PTEN-induced kinase 1 (PINK1)-dependent polyubiquitination of multiple proteins on mitochondrial outer membranes, resulting in the removal of ...defective mitochondria via mitophagy. Because Parkin mutations occur in Parkinson’s disease, a condition associated with the death of dopaminergic neurons in the midbrain, wild-type Parkin is thought to promote neuronal survival. However, here we show that wild-type Parkin greatly sensitized toward apoptosis induced by mitochondrial depolarization but not by proapoptotic stimuli that failed to activate Parkin. Parkin-dependent apoptosis required PINK1 and was efficiently blocked by prosurvival members of the Bcl-2 family or knockdown of Bax and Bak. Upon mitochondrial depolarization, the Bcl-2 family member Mcl-1 underwent rapid Parkin- and PINK1-dependent polyubiquitination and degradation, which sensitized toward apoptosis via opening of the Bax/Bak channel. These data suggest that similar to other sensors of cell stress, such as p53, Parkin has cytoprotective (mitophagy) or cytotoxic modes (apoptosis), depending on the degree of mitochondrial damage.
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•Parkin activation can result in mitophagy or apoptosis•Bcl-2 family proteins regulate Parkin-dependent apoptosis•Parkin and PINK1 promote Mcl-1 degradation•Parkin sensitizes toward apoptosis in response to mitochondrial impairment
Parkin, the ubiquitin ligase that is mutated in Parkinson’s disease, regulates the clearance of damaged mitochondria via selective autophagy (mitophagy). Here, Carroll et al. show that Parkin activation in response to mitochondrial impairment can also result in the elimination of cells containing depolarized mitochondria via apoptosis.
Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of ...long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation.
Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving ...metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.
Dendritic cells play a key role in processing and presenting antigens to naïve T cells to prime adaptive immunity. Circadian rhythms are known to regulate many aspects of immunity; however, the role ...of circadian rhythms in dendritic cell function is still unclear. Here, we show greater T cell responses when mice are immunised in the middle of their rest versus their active phase. We find a circadian rhythm in antigen processing that correlates with rhythms in both mitochondrial morphology and metabolism, dependent on the molecular clock gene, Bmal1. Using Mdivi-1, a compound that promotes mitochondrial fusion, we are able to rescue the circadian deficit in antigen processing and mechanistically link mitochondrial morphology and antigen processing. Furthermore, we find that circadian changes in mitochondrial Ca
are central to the circadian regulation of antigen processing. Our results indicate that rhythmic changes in mitochondrial calcium, which are associated with changes in mitochondrial morphology, regulate antigen processing.
The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required ...for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.
The inflammasome is a cytoplasmic protein complex that processes interleukins (IL)-1β and IL-18, and drives a form of cell death known as pyroptosis. Oligomerization of this complex is actually the ...second step of activation, and a priming step must occur first. This involves transcriptional upregulation of pro-IL-1β, inflammasome sensor NLRP3, or the non-canonical inflammasome sensor caspase-11. An additional aspect of priming is the post-translational modification of particular inflammasome constituents. Priming is typically accomplished in vitro using a microbial Toll-like receptor (TLR) ligand. However, it is now clear that inflammasomes are activated during the progression of sterile inflammatory diseases such as atherosclerosis, metabolic disease, and neuroinflammatory disorders. Therefore, it is time to consider the endogenous factors and mechanisms that may prime the inflammasome in these conditions.
A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the ...functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1β and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1
–/– macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1β. The excessive prooxidant and proinflammatory phenotype of Bmal1
–/– macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1β are known to play a role.
Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created ...by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4-1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-XL, and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rγ⁻/⁻ mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.
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