Showing papers in "The EMBO Journal in 2018"

Tau protein liquid–liquid phase separation can initiate tau aggregation

Abstract: The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase‐separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain. Droplet‐like tau can also be observed in neurons and other cells. We found that tau droplets become gel‐like in minutes, and over days start to spontaneously form thioflavin‐S‐positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.

The complex relationship between TFEB transcription factor phosphorylation and subcellular localization.

Abstract: The MiT-TFE family of basic helix-loop-helix leucine-zipper transcription factors includes four members: TFEB, TFE3, TFEC, and MITF Originally described as oncogenes, these factors play a major role as regulators of lysosome biogenesis, cellular energy homeostasis, and autophagy. An important mechanism by which these transcription factors are regulated involves their shuttling between the surface of lysosomes, the cytoplasm, and the nucleus. Such dynamic changes in subcellular localization occur in response to nutrient fluctuations and various forms of cell stress and are mediated by changes in the phosphorylation of multiple conserved amino acids. Major kinases responsible for MiT-TFE protein phosphorylation include mTOR, ERK, GSK3, and AKT In addition, calcineurin de-phosphorylates MiT-TFE proteins in response to lysosomal calcium release. Thus, through changes in the phosphorylation state of MiT-TFE proteins, lysosome function is coordinated with the cellular metabolic state and cellular demands. This review summarizes the evidence supporting MiT-TFE regulation by phosphorylation at multiple key sites. Elucidation of such regulatory mechanisms is of fundamental importance to understand how these transcription factors contribute to both health and disease.

Rab29 activation of the Parkinson's disease‐associated LRRK2 kinase

Abstract: Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector-binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing

Abstract: TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer9s disease Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD) Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro , fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation

Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

Abstract: During apoptosis, pro-apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP Such caspase-independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS-STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS-STING signalling pathway. Using super-resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP In a temporal manner, we find that following MOMP, BAX/BAK-mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK-dependent MOMP Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase-independent cell death.

Attenuation of cGAS-STING Signaling Is Mediated by a p62/SQSTM1-dependent Autophagy Pathway Activated by TBK1

Abstract: Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

p62 filaments capture and present ubiquitinated cargos for autophagy

Abstract: The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin-proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62-dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross-linked by the substrates. The reaction is inhibited by free ubiquitin, K48-, and K63-linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy.

Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution.

Abstract: In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho-GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double-knockout mouse embryos and single- and double-knockout embryonic fibroblasts, we demonstrate the essential and non-redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double-knockout cells. In contrast, we show that TRAK2-mediated retrograde mitochondrial transport is Miro1-dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin-dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double-knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine-tune actin- and tubulin-dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments

Abstract: Proteolytic removal of membrane protein ectodomains (ectodomain shedding) is a post-translational modification that controls levels and function of hundreds of membrane proteins. The contributing proteases, referred to as sheddases, act as important molecular switches in processes ranging from signaling to cell adhesion. When deregulated, ectodomain shedding is linked to pathologies such as inflammation and Alzheimer's disease. While proteases of the "a disintegrin and metalloprotease" (ADAM) and "beta-site APP cleaving enzyme" (BACE) families are widely considered as sheddases, in recent years a much broader range of proteases, including intramembrane and soluble proteases, were shown to catalyze similar cleavage reactions. This review demonstrates that shedding is a fundamental process in cell biology and discusses the current understanding of sheddases and their substrates, molecular mechanisms and cellular localizations, as well as physiological functions of protein ectodomain shedding. Moreover, we provide an operational definition of shedding and highlight recent conceptual advances in the field. While new developments in proteomics facilitate substrate discovery, we expect that shedding is not a rare exception, but rather the rule for many membrane proteins, and that many more interesting shedding functions await discovery.

ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival.

Abstract: Although lysosomes perform a number of essential cellular functions, damaged lysosomes represent a potential hazard to the cell. Such lysosomes are therefore engulfed by autophagic membranes in the process known as lysophagy, which is initiated by recognition of luminal glycoprotein domains by cytosolic lectins such as Galectin‐3. Here, we show that, under various conditions that cause injury to the lysosome membrane, components of the endosomal sorting complex required for transport (ESCRT)‐I, ESCRT‐II, and ESCRT‐III are recruited. This recruitment occurs before that of Galectin‐3 and the lysophagy machinery. Subunits of the ESCRT‐III complex show a particularly prominent recruitment, which depends on the ESCRT‐I component TSG101 and the TSG101‐ and ESCRT‐III‐binding protein ALIX. Interference with ESCRT recruitment abolishes lysosome repair and causes otherwise reversible lysosome damage to become cell lethal. Vacuoles containing the intracellular pathogen Coxiella burnetii show reversible ESCRT recruitment, and interference with this recruitment reduces intravacuolar bacterial replication. We conclude that the cell is equipped with an endogenous mechanism for lysosome repair which protects against lysosomal damage‐induced cell death but which also provides a potential advantage for intracellular pathogens.

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Abstract: Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

The assembly of lipid droplets and their roles in challenged cells

Abstract: Cytoplasmic lipid droplets are important organelles in nearly every eukaryotic and some prokaryotic cells. Storing and providing energy is their main function, but they do not work in isolation. They respond to stimuli initiated either on the cell surface or in the cytoplasm as conditions change. Cellular stresses such as starvation and invasion are internal insults that evoke changes in droplet metabolism and dynamics. This review will first outline lipid droplet assembly and then discuss how droplets respond to stress and in particular nutrient starvation. Finally, the role of droplets in viral and microbial invasion will be presented, where an unresolved issue is whether changes in droplet abundance promote the invader, defend the host, to try to do both. The challenges of stress and infection are often accompanied by changes in physical contacts between droplets and other organelles. How these changes may result in improving cellular physiology, an ongoing focus in the field, is discussed.

The WD40 domain of ATG16L1 is required for its non‐canonical role in lipidation of LC3 at single membranes

Abstract: A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.

Mechanism of membrane pore formation by human gasdermin-D

Abstract: Gasdermin‐D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N‐terminal domain (GSDMD Nterm ) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMD Nterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high‐resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMD Nterm inserts and assembles in membranes. We observe GSDMD Nterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMD Nterm assembles arc‐, slit‐, and ring‐shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase‐1, caspase‐4, or caspase‐5. Using time‐lapse AFM, we monitor how GSDMD Nterm assembles into arc‐shaped oligomers that can transform into larger slit‐shaped and finally into stable ring‐shaped oligomers. Our observations translate into a mechanistic model of GSDMD Nterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression

Abstract: Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo. Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti‐angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor‐α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.

LPS targets host guanylate‐binding proteins to the bacterial outer membrane for non‐canonical inflammasome activation

Abstract: Pathogenic and commensal Gram‐negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase‐11 non‐canonical inflammasome. Here, we show that OMV‐induced inflammasome activation requires TLR4‐TRIF signaling, the production of type I interferons, and the action of guanylate‐binding proteins (GBPs), both in macrophages and in vivo . Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram‐negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell‐autonomous immunity and innate immunity.

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Abstract: Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.

Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency.

Abstract: Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle-specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF-κB activation, and enhanced expression of pro-inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF-κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell-autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF-κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.

Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

Abstract: Retromer is an endosomal multi-protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans-Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer-associated RAB7-specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer-dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin-mediated mitophagy.

Regenerative proliferation of differentiated cells by mTORC1‐dependent paligenosis

Abstract: In 1900, Adami speculated that a sequence of context-independent energetic and structural changes governed the reversion of differentiated cells to a proliferative, regenerative state. Accordingly, we show here that differentiated cells in diverse organs become proliferative via a shared program. Metaplasia-inducing injury caused both gastric chief and pancreatic acinar cells to decrease mTORC1 activity and massively upregulate lysosomes/autophagosomes; then increase damage associated metaplastic genes such as Sox9; and finally reactivate mTORC1 and re-enter the cell cycle. Blocking mTORC1 permitted autophagy and metaplastic gene induction but blocked cell cycle re-entry at S-phase. In kidney and liver regeneration and in human gastric metaplasia, mTORC1 also correlated with proliferation. In lysosome-defective Gnptab-/- mice, both metaplasia-associated gene expression changes and mTORC1-mediated proliferation were deficient in pancreas and stomach. Our findings indicate differentiated cells become proliferative using a sequential program with intervening checkpoints: (i) differentiated cell structure degradation; (ii) metaplasia- or progenitor-associated gene induction; (iii) cell cycle re-entry. We propose this program, which we term "paligenosis", is a fundamental process, like apoptosis, available to differentiated cells to fuel regeneration following injury.

Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis.

Abstract: Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non‐structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11‐linked poly‐ubiquitin chains from caspase‐1 at Lys134, thus inhibiting the proteasomal degradation of caspase‐1. The enhanced stabilization of caspase‐1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKV in vitro and in vivo . Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future.

ER‐to‐lysosome‐associated degradation of proteasome‐resistant ATZ polymers occurs via receptor‐mediated vesicular transport

Abstract: Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.

PIF4-induced BR synthesis is critical to diurnal and thermomorphogenic growth.

Abstract: The Arabidopsis PIF4 and BES1/BZR1 transcription factors antagonize light signaling by facilitating co‐activated expression of a large number of cell wall‐loosening and auxin‐related genes. While PIF4 directly activates expression of these targets, BES1 and BZR1 activity switch from a repressive to an activator function, depending on interaction with TOPLESS and other families of regulators including PIFs. However, the complexity of this regulation and its role in diurnal control of plant growth and brassinosteroid (BR) levels is little understood. We show by using a protein array that BES1, PIF4, and the BES1‐PIF4 complex recognize different DNA elements, thus revealing a distinctive cis‐regulatory code beneath BES1‐repressive and PIF4 co‐activation function. BES1 homodimers bind to conserved BRRE‐ and G‐box elements in the BR biosynthetic promoters and inhibit their expression during the day, while elevated PIF4 competes for BES1 homodimer formation, resulting in de‐repressed BR biosynthesis at dawn and in response to warmth. Our findings demonstrate a central role of PIF4 in BR synthesis activation, increased BR levels being essential to thermomorphogenic hypocotyl growth.

Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response.

Abstract: Elevated expression of RNA binding protein HNRNPC has been reported in cancer cells, while the essentialness and functions of HNRNPC in tumors were not clear. We showed that repression of HNRNPC in the breast cancer cells MCF7 and T47D inhibited cell proliferation and tumor growth. Our computational inference of the key pathways and extensive experimental investigations revealed that the cascade of interferon responses mediated by RIG‐I was responsible for such tumor‐inhibitory effect. Interestingly, repression of HNRNPC resulted in accumulation of endogenous double‐stranded RNA (dsRNA), the binding ligand of RIG‐I. These up‐regulated dsRNA species were highly enriched by Alu sequences and mostly originated from pre‐mRNA introns that harbor the known HNRNPC binding sites. Such source of dsRNA is different than the recently well‐characterized endogenous retroviruses that encode dsRNA. In summary, essentialness of HNRNPC in the breast cancer cells was attributed to its function in controlling the endogenous dsRNA and the down‐stream interferon response. This is a novel extension from the previous understandings about HNRNPC in binding with introns and regulating RNA splicing.

Mice with endogenous TDP‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

Abstract: TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

OST1-mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses.

Abstract: Cold stress is a major environmental factor that negatively affects plant growth and survival. OST1 has been identified as a key protein kinase in plant response to cold stress; however, little is known about the underlying molecular mechanism. In this study, we identified BTF3 and BTF3L (BTF3‐like), β‐subunits of a nascent polypeptide‐associated complex (NAC), as OST1 substrates that positively regulate freezing tolerance. OST1 phosphorylates BTF3 and BTF3L in vitro and in vivo , and facilitates their interaction with C‐repeat‐binding factors (CBFs) to promote CBF stability under cold stress. The phosphorylation of BTF3L at the Ser50 residue by OST1 is required for its function in regulating freezing tolerance. In addition, BTF3 and BTF3L proteins positively regulate the expression of CBF genes. These findings unravel a molecular mechanism by which OST1‐BTF3‐CBF module regulates plant response to cold stress.

LRRC25 inhibits type I IFN signaling by targeting ISG15-associated RIG-I for autophagic degradation.

Abstract: The RIG-I-like receptors (RLRs) are critical for protection against RNA virus infection, and their activities must be stringently controlled to maintain immune homeostasis. Here, we report that leucine-rich repeat containing protein 25 (LRRC25) is a key negative regulator of RLR-mediated type I interferon (IFN) signaling. Upon RNA virus infection, LRRC25 specifically binds to ISG15-associated RIG-I to promote interaction between RIG-I and the autophagic cargo receptor p62 and to mediate RIG-I degradation via selective autophagy. Depletion of either LRRC25 or ISG15 abrogates RIG-I-p62 interaction as well as the autophagic degradation of RIG-I. Collectively, our findings identify a previously unrecognized role of LRRC25 in type I IFN signaling activation by which LRRC25 acts as a secondary receptor to assist RIG-I delivery to autophagosomes for degradation in a p62-dependent manner.

NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS.

Abstract: Astrocytes are involved in non‐cell‐autonomous pathogenic cascades in amyotrophic lateral sclerosis (ALS); however, their role is still debated. We show that astrocytic NF‐κB activation drives microglial proliferation and leukocyte infiltration in the SOD1 (G93A) ALS model. This response prolongs the presymptomatic phase, delaying muscle denervation and decreasing disease burden, but turns detrimental in the symptomatic phase, accelerating disease progression. The transition corresponds to a shift in the microglial phenotype showing two effects that can be dissociated by temporally controlling NF‐κB activation. While NF‐κB activation in astrocytes induced a Wnt‐dependent microglial proliferation in the presymptomatic phase with neuroprotective effects on motoneurons, in later stage, astrocyte NF‐κB‐dependent microglial activation caused an accelerated disease progression. Notably, suppression of the early microglial response by CB2R agonists had acute detrimental effects. These data identify astrocytes as important regulators of microglia expansion and immune response. Therefore, stage‐dependent microglia modulation may be an effective therapeutic strategy in ALS.

Infiltrative and drug-resistant slow-cycling cells support metabolic heterogeneity in glioblastoma.

Abstract: Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast‐cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow‐cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose‐deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7‐related metabolic pathways is a viable therapeutic strategy.

Queuosine-modified tRNAs confer nutritional control of protein translation.

Abstract: Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q-tRNA levels promote Dnmt2-mediated methylation of tRNA Asp and control translational speed of Q-decoded codons as well as at near-cognate codons. Deregulation of translation upon queuine depletion results in unfolded proteins that trigger endoplasmic reticulum stress and activation of the unfolded protein response, both in cultured human cell lines and in germ-free mice fed with a queuosine-deficient diet. Taken together, our findings comprehensively resolve the role of this anticodon tRNA modification in the context of native protein translation and describe a novel mechanism that links nutritionally determined modification levels to effective polypeptide synthesis and cellular homeostasis.