Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.     

NADPH oxidases contribute to autophagy regulation

Reactive oxygen species (ROS) are emerging as regulators of autophagy in various cellular contexts. There are many cellular sources of ROS in eukaryotic cells. In phagocytes, the critical immune cells for host defense, the Nox2 NADPH oxidase generates ROS during phagocytosis and plays a central role in microbial killing. Toll-like receptors (TLRs) are important membrane microbial sensing receptors, which can activate Nox2,¹ and were recently demonstrated to signal autophagy targeting of phagosomes to promote their maturation.² Our recent study reveals that Nox2 activity and its generated ROS are key signals that induce TLR-activated autophagy of phagosomes. Our results provide the first evidence that ROS from the Nox2 NADPH oxidase can contribute to regulating autophagy in host defense against bacteria. The association of TLR, Nox2 and autophagy with inflammatory bowel disease (IBD) suggests a significant role of this antibacterial pathway in these diseases.     

Directing p53 to induce autophagy

Comment on: Naidu SR, et al. Cell Cycle 2012; 11:2717-28.     

DAMP-mediated autophagy contributes to drug resistance

Damage-associated molecular pattern molecules (DAMPs) are cellularly derived molecules that can initiate and perpetuate immune responses following trauma, ischemia and other types of tissue damage in the absence of pathogenic infection. High mobility group box 1 (HMGB1) is a prototypical DAMP and is associated with the hallmarks of cancer. Recently we found that HMGB1 release after chemotherapy treatment is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia. Overexpression of HMGB1 by gene transfection rendered leukemia cells resistant to cell death; whereas depletion or inhibition of HMGB1 and autophagy by RNA interference or pharmacological inhibitors increased the sensitivity of leukemia cells to chemotherapeutic drugs. HMGB1 release sustains autophagy as assessed by microtubule-associated protein 1 light chain 3 (LC3) lipidation, redistribution of LC3 into cytoplasmic puncta, degradation of p62 and accumulation of autophagosomes and autolysosomes. Moreover, these data suggest a role for HMGB1 in the regulation of autophagy through the PI3KC3-MEKERK: pathway, supporting the notion that HMGB1-induced autophagy promotes tumor resistance to chemotherapy.     

Targeting autophagy to prevent neonatal stroke damage

Cell death due to cerebral ischemia has been attributed to necrosis and apoptosis, but autophagic mechanisms have recently been implicated as well. Using rats exposed to neonatal focal cerebral ischemia, we have shown that lysosomal and autophagic activities are increased in ischemic neurons, and have obtained strong neuroprotection by post-ischemic inhibition of autophagy.     

A systems biology approach to learning autophagy

With its relevance to our understanding of eukaryotic cell function in the normal and disease state, autophagy is an important topic in modern cell biology; yet, few textbooks discuss autophagy beyond a two- or three-sentence summary. Here, we report an undergraduate/graduate class lesson for the in-depth presentation of autophagy using an active learning approach. By our method, students will work in small groups to solve problems and interpret an actual data set describing genes involved in autophagy. The problem-solving exercises and data set analysis will instill within the students a much greater understanding of the autophagy pathway than can be achieved by simple rote memorization of lecture materials; furthermore, the students will gain a general appreciation of the process by which data are interpreted and eventually formed into an understanding of a given pathway. As the data sets used in these class lessons are largely genomic and complementary in content, students will also understand first-hand the advantage of an integrative or systems biology study: No single data set can be used to define the pathway in full-the information from multiple complementary studies must be integrated in order to recapitulate our present understanding of the pathways mediating autophagy. In total, our teaching methodology offers an effective presentation of autophagy as well as a general template for the discussion of nearly any signaling pathway within the eukaryotic kingdom.     

There is more to autophagy than induction

Considerable attention has been paid to the topic of autophagy induction. In part, this is because of the potential for modulating this process for therapeutic purposes. Of course we know that induced autophagy can also be problematic—for example, when trying to eliminate an established tumor that might be relying on autophagy for its own cytoprotective uses. Accordingly, inhibitory mechanisms have been considered; however, the corresponding studies have tended to focus on the pathways that block autophagy under noninducing conditions, such as when nutrients are available. In contrast, relatively little is known about the mechanisms for inhibiting autophagy under inducing conditions. Yet, this type of regulation must be occurring on a routine basis. We know that dysregulation of autophagy, e.g., due to improper activation of Beclin 1 leading to excessive autophagy activity, can cause cell death.¹ Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122:927 - 39; http://dx.doi.org/10.1016/j.cell.2005.07.002; PMID: 16179260 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Accordingly, we assume that during starvation or other inducing conditions there must be a mechanism to modulate autophagy. That is, once you turn it on, you do not want to let it continue unchecked. But how is autophagy downregulated when the inducing conditions still exist?     

The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction

Autophagy is a membrane trafficking process involved in intracellular degradation and recycling in eukaryotic cells. DRAM2 (damage-regulated autophagy modulator 2) is a homologue of DRAM that regulates p53-mediated cell death. As its name implies, DRAM expression induces autophagy in a p53-dependent manner; however, the role of DRAM2 in autophagy is not clear. In this study, we report that DRAM2 expression contributes to autophagy induction. Overexpression of DRAM2 induces cytoplasmic GFP-LC3 punctuates, and increases the level of endogenous LC3-II. Moreover, the silencing of endogenous DRAM2 interferes with starvation-induced autophagy. Thus, we propose that DRAM2 as well as DRAM are involved in autophagy.     

Inflammation and autophagy conspire to promote tumor growth

Comment on: Martinez-Outschoorn UE, et al. Cell Cycle 2011; 10:1784-93.     

MAP1S enhances autophagy to suppress tumorigenesis

Microtubule-associated protein 1 small form (MAP1S; originally named C19ORF5) was identified as serving as linkers to connect mitochondria with microtubules for trafficking, and to bridge the autophagy machinery with microtubules and mitochondria to affect autophagosomal biogenesis and degradation. We found that MAP1S levels become elevated immediately in response to diethylnitrosamine-induced or genome instability-driven metabolic stress in a murine model of hepatocarcinoma. Elevation of MAP1S enhances autophagy to remove p62-associated aggresomes and dysfunctional organelles that trigger DNA double-strand (DSB) breaks and genome instability. The early accumulation of an unstable genome prior to signs of tumorigenesis suggested that genome instability causes tumorigenesis. After tumorigenesis, tumor development then triggers the activation of autophagy to reduce genome instability in tumor foci. We concluded that an increase in MAP1S levels triggers autophagy in order to suppress genome instability so that both the incidence of diethylnitrosamine-induced hepatocarcinogenesis and malignant progression are suppressed. Thus, a link between MAP1S-enhanced autophagy and suppression of genomic instability and tumorigenesis has been established.     

Spermidine boosts autophagy to protect from synapse aging

All animals form memories to adapt their behavior in a context-dependent manner. With increasing age, however, forming new memories becomes less efficient. While synaptic plasticity promotes memory formation, the etiology of age-induced memory formation remained enigmatic. Previous work showed that simple feeding of polyamine spermidine protects from age-induced memory impairment in Drosophila. Most recent work now shows that spermidine operates directly at synapses, allowing for an autophagy-dependent homeostatic regulation of presynaptic specializations. How exactly autophagic regulations intersect with synaptic plasticity should be an interesting subject for future research.     

Polymorphisms in autophagy genes and susceptibility to tuberculosis

Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB) among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (p = 0.02) and MTOR (p = 0.02) and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (p = 0.04). All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.