Targeted Dephosphorylation of Tau by Phosphorylation Targeting Chimeras (PhosTACs) as a Therapeutic Modality.

Microtubule-associated protein tau is essential for microtubule assembly and stabilization. Hyperphosphorylation of the microtubule-associated protein tau plays an important pathological role in the development of Alzheimer's disease and other tauopathies. In vivo studies using kinase inhibitors suggest that reducing tau phosphorylation levels has therapeutic potential; however, such approaches showed limited benefits. We sought to further develop our phosphorylation targeting chimera (PhosTAC) technology to specifically induce tau dephosphorylation. Herein, we use small molecule-based PhosTACs to recruit tau to PP2A, a native tau phosphatase. PhosTACs induced the formation of a stable ternary complex, leading to rapid, efficient, and sustained tau dephosphorylation, which also correlated with the enhanced downregulation of tau protein. Mass spectrometry data validated that PhosTACs downregulated multiple phosphorylation sites of tau. We believe that PhosTAC possesses several advantages over current strategies to modulate tau phosphorylation and represents a new avenue for disease-modifying therapies for tauopathies.

Targeted Degradation of mRNA Decapping Enzyme DcpS by a VHL-Recruiting PROTAC.

The RNA decapping scavenger protein, DcpS, has recently been identified as a dependency in acute myeloid leukemia (AML). The potent DcpS inhibitor RG3039 attenuates AML cell viability, and shRNA knockdown of DcpS is also antiproliferative. Importantly, DcpS was found to be non-essential in normal human hematopoietic cells, which opens a therapeutic window for AML treatment by DcpS modulation. Considering this strong DcpS dependence in AML cell lines, we explored PROTAC-mediated degradation as an alternative strategy to modulate DcpS activity. Herein, we report the development of JCS-1, a PROTAC exhibiting effective degradation of DcpS at nanomolar concentrations. JCS-1 non-covalently binds DcpS with a RG3039-based warhead and recruits the E3 ligase VHL, which induces potent, rapid, and sustained DcpS degradation in several AML cell lines. JCS-1 serves as a chemical biology tool to interrogate DcpS degradation and associated changes in RNA processes in different cellular contexts, which may be an attractive strategy for the treatment of AML and other DcpS-dependent genetic disorders.

Migration-induced cell shattering due to DOCK8 deficiency causes a type 2-biased helper T cell response.

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

The plot thickens: OTULIN regulation in cell death.

We recently demonstrated that post-translational modifications of the OTU deubiquitinase with linear linkage specificity (OTULIN) regulate its function in cell death. OTULIN hyper-phosphorylation promotes necroptosis by locking ring finger protein 31 (RNF31, also known as HOIP) away from the cylindromatosis (CYLD) complex, resulting in altered receptor interacting serine/threonine kinase 1 (RIPK1) ubiquitination. Further, we identified dual specificity phosphatase 14 (DUSP14) as an OTULIN phosphatase that limits necroptosis.

Cross-regulation between LUBAC and caspase-1 modulates cell death and inflammation.

The linear ubiquitin assembly complex (LUBAC) is an essential component of the innate and adaptive immune system. Modification of cellular substrates with linear polyubiquitin chains is a key regulatory step in signal transduction that impacts cell death and inflammatory signaling downstream of various innate immunity receptors. Loss-of-function mutations in the LUBAC components HOIP and HOIL-1 yield a systemic autoinflammatory disease in humans, whereas their genetic ablation is embryonically lethal in mice. Deficiency of the LUBAC adaptor protein Sharpin results in a multi-organ inflammatory disease in mice characterized by chronic proliferative dermatitis (cpdm), which is propagated by TNFR1-induced and RIPK1-mediated keratinocyte cell death. We have previously shown that caspase-1 and -11 promoted the dermatitis pathology of cpdm mice and mediated cell death in the skin. Here, we describe a reciprocal regulation of caspase-1 and LUBAC activities in keratinocytes. We show that LUBAC interacted with caspase-1 via HOIP and modified its CARD domain with linear polyubiquitin and that depletion of HOIP or Sharpin resulted in heightened caspase-1 activation and cell death in response to inflammasome activation, unlike what is observed in macrophages. Reciprocally, caspase-1, as well as caspase-8, regulated LUBAC activity by proteolytically processing HOIP at Asp-348 and Asp-387 during the execution of cell death. HOIP processing impeded substrate ubiquitination in the NF-κB pathway and resulted in enhanced apoptosis. These results highlight a regulatory mechanism underlying efficient apoptosis in keratinocytes and provide further evidence of a cross-talk between inflammatory and cell death pathways.

Post-translational Modification of OTULIN Regulates Ubiquitin Dynamics and Cell Death.

Linear ubiquitination has emerged as an important post-translational modification that regulates NF-κB activation, inflammation, and cell death in both immune and non-immune compartments, including the skin. The deubiquitinase OTULIN specifically disassembles linear ubiquitin chains generated by the linear ubiquitin assembly complex (LUBAC) and is necessary to prevent embryonic lethality and autoinflammatory disease. Here, we dissect the direct role of OTULIN in cell death and find that OTULIN limits apoptosis and necroptosis in keratinocytes. During apoptosis, OTULIN is cleaved by capase-3 at Asp-31 into a C-terminal fragment that restricts caspase activation and cell death. During necroptosis, OTULIN is hyper-phosphorylated at Tyr-56, which modulates RIPK1 ubiquitin dynamics and promotes cell death. OTULIN Tyr-56 phosphorylation is counteracted by the activity of dual-specificity phosphatase 14 (DUSP14), which we identify as an OTULIN phosphatase that limits necroptosis. Our data provide evidence of dynamic post-translational modifications of OTULIN and highlight their importance in cell death outcome.

The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson's disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check.

The Interleukin (IL)-1R1 pathway is a critical negative regulator of PyMT-mediated mammary tumorigenesis and pulmonary metastasis.

Breast cancer is the most common cancer in women and the second leading cause of female cancer-related deaths worldwide. Inflammation is an established hallmark of tumorigenesis and an important determinant of tumor outcome and response to therapy. With advances in cancer immunotherapy, there is an urgent need to dissect the contribution of specific immune effectors in cancer development. Here, we genetically investigated the role of the Interleukin-1 (IL-1) receptor 1 (IL-1R1) pathway in breast cancer tumorigenesis and metastasis using the MMTV-PyMT mouse model. Our results indicate that IL-1R1 signaling suppresses mammary tumor cell proliferation early in tumorigenesis and curbs breast cancer outgrowth and pulmonary metastasis. We show that PyMT/Il1r1-/- mice had a higher primary tumor burden and increased mortality rate compared with IL-1R1-sufficient PyMT control mice. This phenotype was independent of the inflammatory caspases-1/-11 but driven by IL-1α, as PyMT/Il1a-/- mice phenocopied PyMT/Il1r1-/- mice. Collectively, our results suggest that IL-1α-mediated IL-1R1 signaling is tumor-suppressive in PyMT-driven breast cancer.

Caspase-12, but Not Caspase-11, Inhibits Obesity and Insulin Resistance.

Inflammation is well established to significantly impact metabolic diseases. The inflammatory protease caspase-1 has been implicated in metabolic dysfunction; however, a potential role for the related inflammatory caspases is currently unknown. In this study, we investigated a role for caspase-11 and caspase-12 in obesity and insulin resistance. Loss of caspase-12 in two independently generated mouse strains predisposed mice to develop obesity, metabolic inflammation, and insulin resistance, whereas loss of caspase-11 had no effect. The use of bone marrow chimeras determined that deletion of caspase-12 in the radio-resistant compartment was responsible for this metabolic phenotype. The Nlrp3 inflammasome pathway mediated the metabolic syndrome of caspase-12-deficient mice as ablation of Nlrp3 reversed Casp12(-/-) mice obesity phenotype. Although the majority of people lack a functional caspase-12 because of a T(125) single nucleotide polymorphism that introduces a premature stop codon, a fraction of African descendents express full-length caspase-12. Expression of caspase-12 was linked to decreased systemic and adipose tissue inflammation in a cohort of African American obese children. However, analysis of the Dallas Heart Study African American cohort indicated that the coding T(125)C single nucleotide polymorphism was not associated with metabolic parameters in humans, suggesting that host-specific differences mediate the expressivity of metabolic disease.

The Nlrp3 Inflammasome Suppresses Colorectal Cancer Metastatic Growth in the Liver by Promoting Natural Killer Cell Tumoricidal Activity.

The crosstalk between inflammation and tumorigenesis is now clearly established. However, how inflammation is elicited in the metastatic environment and the corresponding contribution of innate immunity pathways in suppressing tumor growth at secondary sites are poorly understood. Here, we show that mice deficient in Nlrp3 inflammasome components had exacerbated liver colorectal cancer metastatic growth, which was mediated by impaired interleukin-18 (IL-18) signaling. Control of tumor growth was independent of differential cancer cell colonization or proliferation, intestinal microbiota effects, or tumoricidal activity by the adaptive immune system. Instead, the inflammasome-IL-18 pathway impacted maturation of hepatic NK cells, surface expression of the death ligand FasL, and capacity to kill FasL-sensitive tumors. Our results define a regulatory signaling circuit within the innate immune system linking inflammasome activation to effective NK-cell-mediated tumor attack required to suppress colorectal cancer growth in the liver.

The Inflammatory Caspases-1 and -11 Mediate the Pathogenesis of Dermatitis in Sharpin-Deficient Mice.

Chronic proliferative dermatitis in mice (cpdm) is a spontaneous multiorgan inflammatory disorder with pathological hallmarks similar to atopic dermatitis and psoriasis in humans. Cpdm mice lack expression of SHANK-associated RH domain-interacting protein, an adaptor of the linear ubiquitin assembly complex, which acts in the NF-κB pathway to promote inflammation and protect from apoptosis and necroptosis. Although skin inflammation in cpdm mice is driven by TNF- and RIPK1-induced cell death, the contribution of initiating innate immunity sensors and additional inflammatory pathways remains poorly characterized. In this article, we show that inflammasome signaling, including the expression and activation of the inflammatory caspase-1 and -11 and IL-1 family cytokines, was highly upregulated in the skin of cpdm mice prior to overt disease onset. Genetic ablation of caspase-1 and -11 from cpdm mice significantly reduced skin inflammation and delayed disease onset, whereas systemic immunological disease persisted. Loss of Nlrp3 also attenuated skin disease, albeit more variably. Strikingly, induction of apoptosis and necroptosis effectors was sharply decreased in the absence of caspase-1 and -11. These results position the inflammasome as an important initiating signal in skin disease pathogenesis and provide novel insights about inflammasome and cell death effector cross-talk in the context of inflammatory diseases.

Role of programmed necrosis and cell death in intestinal inflammation.

PURPOSE OF REVIEW: To critically review recent advances on the role of programmed necrosis and other cell death modalities in intestinal health and inflammatory bowel disease. RECENT FINDINGS: Tight regulation of intestinal epithelial cell proliferation and cell death is required for intestinal physiology, and to maintain an integral barrier that restricts microbiota translocation and ensures immune tolerance. Apoptosis has long been considered as a normal part of intestinal epithelial cell turnover. However, recent studies have demonstrated that excessive cell death leads to deleterious intestinal inflammation, as is observed in inflammatory bowel disease. Additionally, a novel form of cell death dubbed programmed necrosis, or necroptosis, has been recently shown to be pathological in the gut. SUMMARY: The role of cell death in the intestine is complex and its potential implication in intestinal diseases, and inflammatory bowel disease in particular, needs to be reevaluated.

Advances in Nod-like receptors (NLR) biology.

The innate immune system is composed of a wide repertoire of conserved pattern recognition receptors (PRRs) able to trigger inflammation and host defense mechanisms in response to endogenous or exogenous pathogenic insults. Among these, nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are intracellular sentinels of cytosolic sanctity capable of orchestrating innate immunity and inflammatory responses following the perception of noxious signals within the cell. In this review, we elaborate on recent advances in the signaling mechanisms of NLRs, operating within inflammasomes or through alternative inflammatory pathways, and discuss the spectrum of their effector functions in innate immunity. We describe the progressive characterization of each NLR with associated controversies and cutting edge discoveries.