ORMDL3 regulates NLRP3 inflammasome activation by maintaining ER-mitochondria contacts in human macrophages and dictates ulcerative colitis patient outcome.

Genome-wide association studies in inflammatory bowel disease have identified risk loci in the orosomucoid-like protein 3/ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene to confer susceptibility to ulcerative colitis (UC), but the underlying functional relevance remains unexplored. Here, we found that a subpopulation of the UC patients who had higher disease activity shows enhanced expression of ORMDL3 compared to the patients with lower disease activity and the non-UC controls. We also found that the patients showing high ORMDL3 mRNA expression have elevated interleukin-1ß cytokine levels indicating positive correlation. Further, knockdown of ORMDL3 in the human monocyte-derived macrophages resulted in significantly reduced interleukin-1ß release. Mechanistically, we report for the first time that ORMDL3 contributes to a mounting inflammatory response via modulating mitochondrial morphology and activation of the NLRP3 inflammasome. Specifically, we observed an increased fragmentation of mitochondria and enhanced contacts with the endoplasmic reticulum (ER) during ORMDL3 over-expression, enabling efficient NLRP3 inflammasome activation. We show that ORMDL3 that was previously known to be localized in the ER also becomes localized to mitochondria-associated membranes and mitochondria during inflammatory conditions. Additionally, ORMDL3 interacts with mitochondrial dynamic regulating protein Fis-1 present in the mitochondria-associated membrane. Accordingly, knockdown of ORMDL3 in a dextran sodium sulfate -induced colitis mouse model showed reduced colitis severity. Taken together, we have uncovered a functional role for ORMDL3 in mounting inflammation during UC pathogenesis by modulating ER-mitochondrial contact and dynamics.

Foot-and-mouth disease virus induces PERK-mediated autophagy to suppress the antiviral interferon response.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in cloven-hoofed animals. FMDV replication-associated viral protein expression induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), in turn inducing autophagy to restore cellular homeostasis. We observed that inhibition of BiP (also known as HSPA5 and GRP78), a master regulator of ER stress and UPR, decreased FMDV infection confirming their involvement. Further, we show that the FMDV infection induces UPR mainly through the PKR-like ER kinase (PERK; also known as EIF2AK3)-mediated pathway. Knockdown of PERK and chemical inhibition of PERK activation resulted in decreased expression of FMDV proteins along with the reduction of autophagy marker protein LC3B-II [the lipidated form of LC3B (also known as MAP1LC3B)]. There are conflicting reports on the role of autophagy in FMDV multiplication. Our study systematically demonstrates that during FMDV infection, PERK-mediated UPR stimulated an increased level of endogenous LC3B-II and turnover of SQSTM1, thus confirming the activation of functional autophagy. Modulation of the UPR and autophagy by pharmacological and genetic approaches resulted in reduced numbers of viral progeny, by enhancing the antiviral interferon response. Taken together, this study underscores the prospect of exploring PERK-mediated autophagy as an antiviral target.

Xenophagy in cancer.

Macroautophagy (herein autophagy) is an intracellular pathway in which cytoplasmic components are captured by double-membrane vesicles (autophagosomes) that eventually fuse with lysosomes to degrade the cargo. Basal levels of autophagy in all eukaryotic cells maintain cellular homeostasis and under conditions of stress, organelles and proteins not essential for survival are degraded. Apart from these functions, cargoes like aggregated proteins, damaged organelles and intracellular pathogens, which are otherwise harmful to cells, are also selectively captured by autophagy and are destined for degradation. In terms of infectious diseases, pathogens are cleared by a specific form of autophagy known as xenophagy. This lysosomal mediated degradation of pathogens also increases the antigen presentation of cells thereby inducing a further immune response. The process of xenophagy provides a broad spectrum of defense mechanism to capture bacterial, viral and protozoan pathogens. However, pathogens have developed ingenious mechanisms to modulate xenophagy to enhance their intracellular survival. Meanwhile, certain pathogens also induce deleterious effects such as chronic inflammation and overexpression of oncogenes in the host system. This over time can increase the susceptibility of the host for tumorigenesis. Hence targeting tumor through anti-microbial mechanisms like xenophagy could be a novel strategy for combinatorial anti-cancer therapy. The recent developments in understanding the role of xenophagy in combating cancer causing pathogens will be discussed in this review.

Vancomycin Derivative Inactivates Carbapenem-Resistant Acinetobacter baumannii and Induces Autophagy.

Vancomycin is a standard drug for the treatment of multidrug-resistant Gram-positive bacterial infections. Albeit, development of resistance (VRE, VRSA) and its inefficacy against persistent infections is a demerit. It is also intrinsically inactive against Gram-negative bacteria. Herein, we report a vancomycin derivative, VanQAmC10, that addresses these challenges. VanQAmC10 was rapidly bactericidal against carbapenem-resistant A. baumannii (6 log10 CFU/mL reduction in 6 h), disrupted A. baumannii biofilms, and eradicated their stationary phase cells. In MRSA infected macrophages, the compound reduced the bacterial burden by 1.3 log10 CFU/mL while vancomycin exhibited a static effect. Further investigation indicated that the compound, unlike vancomycin, promoted the intracellular degradative mechanism, autophagy, in mammalian cells, which may have contributed to its intracellular activity. The findings of the work provide new perspectives on the field of glycopeptide antibiotics.

Restriction of intracellular Salmonella replication by restoring TFEB-mediated xenophagy.

Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular Salmonella typhimurium infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target Salmonella-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down Salmonella replication in SCVs. Acacetin is effective as a xenophagy compound in an in vivo mouse model of infection and reduces intracellular Salmonella burden. ABBREVIATIONS: 3-MA: 3-methyladenine; BafA1: bafilomycin A1; CFU: colony-forming units; DQ-BSA: dye quenched-bovine serum albumin; EEA1: early endosome antigen 1; FITC: fluorescein isothiocyanate; FM 4-64: pyridinium,4-(6-[4-{diethylamino}phenyl]-1,3,5-hexatrienyl)-1-(3[triethylammonio] propyl)-dibromide; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; MAPILC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; RFP: red fluorescent protein; SCVs: Salmonella-containing vacuoles; SD: standard deviation; SDS: sodium dodecyl sulfate; SEM: standard mean error; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TFEB: transcription factor EB.

Chemical Biology Strategies to Study Autophagy.

Growing amount of evidence in the last two decades highlight that macroautophagy (generally referred to as autophagy) is not only indispensable for survival in yeast but also equally important to maintain cellular quality control in higher eukaryotes as well. Importantly, dysfunctional autophagy has been explicitly shown to be involved in various physiological and pathological conditions such as cell death, cancer, neurodegenerative, and other diseases. Therefore, modulation and regulation of the autophagy pathway has emerged as an alternative strategy for the treatment of various disease conditions in the recent years. Several studies have shown genetic or pharmacological modulation of autophagy to be effective in treating cancer, clearing intracellular aggregates and pathogens. Understanding and controlling the autophagic flux, either through a genetic or pharmacological approach is therefore a highly promising approach and of great scientific interest as spatiotemporal and cell-tissue-organ level autophagy regulation is not clearly understood. Indeed, chemical biology approaches that identify small molecule effectors of autophagy have thus a dual benefit: the modulators act as tools to study and understand the process of autophagy, and may also have therapeutic potential. In this review, we discuss different strategies that have appeared to screen and identify potent small molecule modulators of autophagy.

Modulation of Autophagy by a Small Molecule Inverse Agonist of ERRα Is Neuroprotective.

Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA), new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR) independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson's disease (PD), XCT 790 exerted neuroprotective effects in the dopaminergic neurons of nigra by inducing autophagy to clear toxic protein aggregates and, in addition, ameliorated motor co-ordination deficits. Using a chemical biology approach, we unrevealed the role of ERRα in regulating autophagy and can be therapeutic target for neurodegeneration.