Zhixun Dou

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34f/f mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34f/f mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34f/f mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.

Gain-of-function p53 mutants co-opt chromatin pathways to drive cancer growth

TP53 (which encodes p53 protein) is the most frequently mutated gene among all human cancers. Prevalent p53 missense mutations abrogate its tumour suppressive function and lead to a ‘gain-of-function’ (GOF) that promotes cancer. Here we show that p53 GOF mutants bind to and upregulate chromatin regulatory genes, including the methyltransferases MLL1 (also known as KMT2A), MLL2 (also known as KMT2D), and acetyltransferase MOZ (also known as KAT6A or MYST3), resulting in genome-wide increases of histone methylation and acetylation. Analysis of The Cancer Genome Atlas shows specific upregulation of MLL1, MLL2, and MOZ in p53 GOF patient-derived tumours, but not in wild-type p53 or p53 null tumours. Cancer cell proliferation is markedly lowered by genetic knockdown of MLL1 or by pharmacological inhibition of the MLL1 methyltransferase complex. Our study reveals a novel chromatin mechanism underlying the progression of tumours with GOF p53, and suggests new possibilities for designing combinatorial chromatin-based therapies for treating individual cancers driven by prevalent GOF p53 mutations.

Autophagy mediates degradation of nuclear lamina

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3–lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3–lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

Class IA PI3K p110β Subunit Promotes Autophagy through Rab5 Small GTPase in Response to Growth Factor Limitation

Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110β catalytic subunit of the class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes and increases its interaction with the small GTPase Rab5. This p110β-Rab5 association maintains Rab5 in its guanosine triphosphate (GTP)-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110β mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110β acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

The class IA phosphatidylinositol 3-kinase p110-β subunit is a positive regulator of autophagy

p110-β associates with the Vps34–Vps15–Beclin 1–Atg14L complex and facilitates generation of PtdIns(3)P to promote autophagy.

Cytoplasmic chromatin triggers inflammation in senescence and cancer

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS–STING (cyclic GMP–AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin–cGAS–STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Inhibition of Protein Degradation Induces Apoptosis through a Microtubule-Associated Protein 1 Light Chain 3-Mediated Activation of Caspase-8 at Intracellular Membranes

ABSTRACT The accumulation of damaged or misfolded proteins, if unresolved, can lead to a detrimental consequence within cells termed proteotoxicity. Since cancerous cells often display elevated protein synthesis and by-product disposal, inhibition of the protein degradation pathways is an emerging approach for cancer therapy. However, the molecular mechanism underlying proteotoxicity remains largely unclear. We show here that inhibition of proteasomal degradation results in an increased oligomerization and activation of caspase-8 on the cytosolic side of intracellular membranes. This enhanced caspase-8 oligomerization and activation are promoted through its interaction with the ubiquitin-binding protein SQSTM1/p62 and the microtubule-associated protein light chain 3 (LC3), which are enriched at intracellular membranes in response to proteotoxic stress. Silencing LC3 by shRNA, or the LC3 mutants defective in membrane localization or p62 interaction fail to induce caspase-8 activation and apoptosis. Our results unveiled a previously unknown mechanism through which disruption of protein homeostasis induces caspase-8 oligomerization, activation, and apoptosis.

TRIM14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-I–like receptor-mediated innate immune response

Significance The innate immune system plays a key role in host defense that involves the detection of microbial components and a series of signaling events that lead to production of interferons and cytokines. Recently, the identification of mitochondrial antiviral-signaling (MAVS) protein placed mitochondria at the forefront of the innate immune response against virus infection. However, how the MAVS signaling complex is assembled and regulated on the mitochondria outer membrane is only partially understood. Here we show that tripartite motif 14 (TRIM14) facilitates the assembly of the MAVS complex assembly. Upon virus infection, TRIM14 recruits NF-κB essential modulator (NEMO) to the MAVS complex via ubiquitin chains. Knockdown of TRIM14 disrupts the MAVS–NEMO association and attenuates the antiviral response. Our results thus provide a missing link in MAVS-mediated innate immune signaling. Innate immunity provides the first line of host defense against invading microbial pathogens. This defense involves retinoic acid-inducible gene-I–like receptors that detect viral RNA and activate the mitochondrial antiviral-signaling (MAVS) protein, an adaptor protein, leading to activation of the innate antiviral immune response. The mechanisms by which the MAVS signalosome assembles on mitochondria are only partially understood. Here, we identify tripartite motif 14 (TRIM14) as a mediator in the immune response against viral infection. TRIM14 localizes to the outer membrane of mitochondria and interacts with MAVS. Upon viral infection, TRIM14 undergoes Lys-63–linked polyubiquitination at Lys-365 and recruits NF-κB essential modulator to the MAVS signalosome, leading to the activation of both the IFN regulatory factor 3 and NF-κB pathways. Knockdown of TRIM14 disrupts the association between NF-κB essential modulator and MAVS and attenuates the antiviral response. Our results indicate that TRIM14 is a component of the mitochondrial antiviral immunity that facilitates the immune response mediated by retinoic acid-inducible gene-I–like receptors.

TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21-deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

MLL1 is essential for the senescence-associated secretory phenotype

Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces SASP-like inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS- or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression.