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Nucleotide-binding and leucine-rich repeat (NLR) proteins are critical intracellular immune receptors in both animals and plants. Perception of pathogen-derived or stress-associated signals induces NLR oligomerization to form multiprotein complexes called inflammasomes in animals or resistosomes in plants to mediate host immune response. Significant progress has been made during the past few years in our understanding of NLR biology, particularly the structural perspective of these two types of NLR-containing complexes. In this article, we review the latest advances in our structural knowledge of how NLR inflammasomes and resistosomes are activated and assembled and how the structural information provides insight into their distinct mechanisms of action. Commonalities and differences between NLR inflammasomes and resistosomes are also discussed.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0–4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches.

The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels activate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallet et. al, 2015) reported that AMPK in yeast,i.e. Snf1, plays a role in short-term downregulation of TORC1 activity upon acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 contributes to glucose starvation-induced short-term TORC1 inactivation primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phosphorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physiologically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells.

Based on its proposed substrate preferences, the ABC transporter, ABCG36/PDR8/PEN3, from the model plant Arabidopsis stands at the cross-road between growth and defence. Recently, ABCG36 was shown to export a few indolic compounds, including the auxin precursor, indole-3-butyric acid (IBA), and to be implicated in the export of the major phytoalexin of Arabidopsis, camalexin, although clear-cut proof of camalexin transport activity is still lacking.Here we provide strong evidence that ABCG36 catalyses the direct, ATP-dependent export of camalexin over the plasma membrane, however, most likely in functional interplay with non-camalexin transporting ABCG isoforms. We identify the leucin-rich repeat receptor-like kinase, Auxin-induced LRR Kinase1 (ALK1/KIN7/QSK1), as a functional kinase to physically interact with and phosphorylate ABCG36. ABCG36 phosphorylation by ALK1 represses unilaterally IBA but not camalexin export leading to a prioritization of ABCG transport toward defense. As a consequence, phospho-dead mutants of ABCG36, like alk1 and abcg36 alleles, are hypersensitive toward infection with the root pathogen, F. oxysporum, caused by elevated fungal progression.Our findings indicate a novel, direct regulatory circuit between a receptor kinase and an ABC transporter determining transporter substrate specificity. It appears that growth and defense balance decisions in plants are performed on the transporter level by means of a reversible phospho-switch.

Human skin functions as a physical barrier, preventing the entry of foreign pathogens while also accommodating a myriad of commensal microorganisms. A key contributor to the skin landscape is the sebaceous gland. Mice devoid of sebocytes are prone to skin infection, yet our understanding of how sebocytes function in host defense is incomplete. Here, we show that the small proline-rich proteins, SPRR1 and SPRR2 are bactericidal in skin. SPRR1B and SPPR2A were induced in human sebocytes by exposure to the bacterial cell wall component lipopolysaccharide (LPS). Colonization of germ-free mice was insufficient to trigger increased SPRR expression in mouse skin, but LPS injected into mouse skin stimulated increased expression of the mouse SPRR orthologous genes, Sprr1a and Sprr2a, through activation of MYD88. Both mouse and human SPRR proteins displayed potent bactericidal activity against MRSA (methicillin-resistant Staphylococcus aureus), Pseudomonas aeruginosa, and skin commensals. Thus, Sprr1a−/−;Sprr2a−/− mice are more susceptible to MRSA and P. aeruginosa skin infection. Lastly, mechanistic studies demonstrate that SPRR proteins exert their bactericidal activity through binding and disruption of the bacterial membrane. Taken together, these findings provide insight into the regulation and antimicrobial function of SPRR proteins in skin and how the skin defends the host against systemic infection.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by TBK1 (Tank-binding kinase 1) activating a LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting expression of pro-inflammatory genes and proteins. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.SummaryAutophagy is well known for its anti-inflammatory effects. Here, we highlight that selective, autophagy-dependent degradation of the inflammation repressor TNIP1 supports pro-inflammatory gene and protein expression. Similarly as in cancer, autophagy appears to play a dual role in controlling inflammation.

Vertebrate lonesome kinase (VLK) is the only known extracellular tyrosine kinase, but its physiological functions are largely unknown. We show that VLK is highly expressed in hepatocytes of neonatal mice, but downregulated during adulthood. To determine the role of VLK in liver homeostasis and regeneration, we generated mice with a hepatocyte-specific knockout of the VLK gene (Pkdcc). Cultured progenitor cells established from primary hepatocytes of Pkdcc knockout mice produced a secretome, which promoted their own proliferation in 3D spheroids and proliferation of cultured fibroblasts. In vivo, Pkdcc knockout mice developed liver steatosis with signs of inflammation and perivascular fibrosis upon aging, combined with expansion of liver progenitor cells. In response to chronic CCl4-induced liver injury, the pattern of deposited collagen was significantly altered in these mice. The liver injury marker alpha-fetoprotein (AFP) was increased in the secretome of VLK-deficient cultured progenitor cells and in liver tissues of aged or CCl4-treated knockout mice. These results support a key role for VLK and extracellular protein phosphorylation in liver homeostasis and repair through paracrine control of liver cell function and regulation of appropriate collagen deposition., Journal of Cell Science, 135 (7), ISSN:1477-9137, ISSN:0021-9533

AMPlifying type 2 immunity Antimicrobial proteins (AMPs) are a frontline defense against pathogenic microorganisms at mucosal surfaces. These cationic molecules inactivate their targets primarily by disrupting cell walls and membranes. Hu et al . found that small proline-rich protein 2A (SPRR2A) is a bactericidal protein produced in the gut that targets Gram-positive bacteria and is phylogenetically distinct from all other known AMPs (see the Perspective by Harris and Wickramasinghe). SPRR2A production is selectively enhanced by type 2 cytokines such as interleukin-4 and -13 that are elicited by helminth infection. Mice lacking SPRR2A are unable to prevent intestinal bacteria from invading the intestinal barrier after helminths damage the intestinal epithelium. SPRR2A is thus a critical component of type 2 immunity that protects against the bacterial invasion and dissemination that follow helminth infection. —STS

SAAving vitamin A–mediated immunity The vitamin A metabolite retinol is critical for B and T cell development and homing to the gut. Intestinal myeloid cells such as dendritic cells and macrophages take up retinol and process it into retinoic acid (RA), which in turn initiates RA-dependent gene expression programs in lymphocytes. Bang et al . identified LDL receptor-related protein 1 (LRP1) as a myeloid cell surface receptor for retinol. LRP1 binds retinol chaperoned by serum amyloid A (SAA) proteins, and SAA–retinol complexes are then endocytosed and metabolized by myeloid cells. Mice lacking either Saa or myeloid-specific Lrp1 exhibited profound impairments in vitamin A–mediated immunity. B and T cell trafficking to the intestine, immunoglobulin A production by B cells, and protection from enteric Salmonella Typhimurium infection were all diminished when either of these crucial players was missing. —STS

The eukaryotic TORC1 kinase assimilates diverse environmental cues, including growth factors and nutrients, to control growth by tuning anabolic and catabolic processes. In yeast, TORC1 stimulates protein synthesis in response to abundant nutrients primarily through its proximal effector kinase Sch9. Conversely, TORC1 inhibition following nutrient limitation unlocks various distally controlled kinases (e.g., Atg1, Gcn2, Npr1, Rim15, Slt2/Mpk1, and Yak1), which cooperate through poorly defined circuits to orchestrate the quiescence program. To better define the signaling landscape of the latter kinases, we use in vivo quantitative phosphoproteomics. Through pinpointing known and uncharted Npr1, Rim15, Slt2/Mpk1, and Yak1 effectors, our study examines the architecture of the distally controlled TORC1 kinase network. Accordingly, this is built on a combination of discrete, convergent, and multilayered feedback regulatory mechanisms, which likely ensure homeostatic control of and/or robust responses by TORC1 and its effector kinases under fluctuating nutritional conditions.

Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9–engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B’s role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD–linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.

Significance Vitamin A is an essential nutrient for immune system development and function. After absorption from the diet, vitamin A is converted to retinol, which is delivered to cells and tissues by retinol-binding proteins. Serum amyloid A (SAA) proteins are retinol-binding proteins that transport retinol specifically during an infection. In this study, we illuminate the molecular details of how SAA proteins bind to retinol. We present the cocrystal structure of mouse SAA3 bound to retinol, which reveals that 3 molecules of SAA3 assemble to form a deep binding pocket that protects retinol, a fat-soluble molecule, from the aqueous environment. Our findings thus provide structural insight into how retinol is transported throughout the body during infection., Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization. In infected mice, SAA proteins circulate in association with the vitamin A derivative retinol, suggesting that SAAs transport retinol during infection. Here we illuminate a structural basis for the retinol–SAA interaction. In the bloodstream of infected mice, most SAA is complexed with high-density lipoprotein (HDL). However, we found that the majority of the circulating retinol was associated with the small fraction of SAA proteins that circulate without binding to HDL, thus identifying free SAA as the predominant retinol-binding form in vivo. We then determined the crystal structure of retinol-bound mouse SAA3 at a resolution of 2.2 Å. Retinol-bound SAA3 formed a novel asymmetric trimeric assembly that was generated by the hydrophobic packing of the conserved amphipathic helices α1 and α3. This hydrophobic packing created a retinol-binding pocket in the center of the trimer, which was confirmed by mutagenesis studies. Together, these findings illuminate the molecular basis for retinol transport by SAA proteins during infection.

Kvainickas et al. establish that retromer, a coat complex that promotes endosomal recycling, is also required for the activation of the lysosomal mTORC1 complex. Mechanistically, retromer and the retromer-associated protein TBC1D5 separate mTORC1 and RAB7 microdomains on late endosomes, which is necessary for amino acid–mediated mTORC1 activation., Retromer is an evolutionarily conserved multiprotein complex that orchestrates the endocytic recycling of integral membrane proteins. Here, we demonstrate that retromer is also required to maintain lysosomal amino acid signaling through mTORC1 across species. Without retromer, amino acids no longer stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. Mechanistically, we show that its effect on mTORC1 activity is not linked to retromer’s role in the recycling of transmembrane proteins. Instead, retromer cooperates with the RAB7-GAP TBC1D5 to restrict late endosomal RAB7 into microdomains that are spatially separated from the amino acid–sensing domains. Upon loss of retromer, RAB7 expands into the ragulator-decorated amino acid–sensing domains and interferes with RAG-GTPase and mTORC1 recruitment. Depletion of retromer in Caenorhabditis elegans reduces mTORC1 signaling and extends the lifespan of the worms, confirming an evolutionarily conserved and unexpected role for retromer in the regulation of mTORC1 activity and longevity.

ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome biogenesis. To uncover if ULK1 has broader functions also regulating subsequent steps of autophagosome turnover, i.e., maturation, lysosomal fusion, and degradation, we performed a set of unbiased phosphoproteomic experiments employing mouse and human cells in combination with genetic and environmental perturbations. We characterized more than 1,000 potential ULK1 target sites of which many affect proteins known to be involved in all phases of the autophagosome life cycle. To better understand which of these 1,000 phosphosites were directly phosphorylated by ULK1, in contrast to downstream kinases being activated or phosphatases being inhibited by ULK1, we developed a proteome-scale in vitro kinase assay and characterized 187 phosphosites on 157 proteins as bona fide ULK1 target sites. Interestingly, our results highlight an intricate crosstalk between ULK1 and protein phosphatases. Focusing on STRN (striatin), a regulatory subunit of PPP2/PP2A (protein phosphatase 2), we identified a positive feedback loop linked to ULK1 and promoting autophagy.

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.

Summary: The evolutionarily conserved ULK1 kinase complex acts as gatekeeper of canonical autophagy and regulates induction of autophagosome biogenesis. To better understand control of ULK1 and analyze whether ULK1 has broader functions that are also linked to the later steps of autophagy, we perform comprehensive phosphoproteomic analyses. Combining in vivo with in vitro data, we identify numerous direct ULK1 target sites within autophagy-relevant proteins that are critical for autophagosome maturation and turnover. In addition, we highlight an intimate crosstalk between ULK1 and several phosphatase complexes. ULK1 is not only a PP2A target but also directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A and serving as positive feedback to promote autophagy-dependent protein turnover. Thus, ULK1 and phosphatase activities are tightly coordinated to robustly regulate protein degradation by autophagy.

Macroautophagy/autophagy is a highly conserved eukaryotic molecular process that facilitates the recycling of superfluous cytoplasmic materials, damaged organelles, and invading pathogens, resulting in proper cellular homeostasis and survival during stress conditions. Autophagy is stringently regulated at multiple stages, including control at transcriptional, translational, and posttranslational levels. In this work, we identified a mechanism by which regulation of autophagy is achieved through the posttranslational modification of Atg9. Here, we show that, in order to limit autophagy to a low, basal level during normal conditions, Atg9 is ubiquitinated and subsequently targeted for degradation in a proteasome-dependent manner through the action of the E3 ligase Met30. When cells require increased autophagy flux to respond to nutrient deprivation, the proteolysis of Atg9 is significantly reduced. Overall, this work reveals an additional layer of mechanistic regulation that allows cells to further maintain appropriate levels of autophagy and to rapidly induce this process in response to stress.

Summary The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the β-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.

Organelles of the endomembrane system maintain their identity and integrity during growth or stress conditions by homeostatic mechanisms that regulate membrane flux and biogenesis. At lysosomes and endosomes, the Fab1 lipid kinase complex and the nutrient-regulated target of rapamycin complex 1 (TORC1) control the integrity of the endolysosomal homeostasis and cellular metabolism. Both complexes are functionally connected as Fab1-dependent generation of PI(3,5)P2 supports TORC1 activity. Here, we identify Fab1 as a target of TORC1 on signaling endosomes, which are distinct from multivesicular bodies, and provide mechanistic insight into their crosstalk. Accordingly, TORC1 can phosphorylate Fab1 proximal to its PI3P-interacting FYVE domain, which causes Fab1 to shift to signaling endosomes, where it generates PI(3,5)P2. This, in turn, regulates (1) vacuole morphology, (2) recruitment of TORC1 and the TORC1-regulatory Rag GTPase-containing EGO complex to signaling endosomes, and (3) TORC1 activity. Thus, our study unravels a regulatory feedback loop between TORC1 and the Fab1 complex that controls signaling at endolysosomes. ispartof: CURRENT BIOLOGY vol:31 issue:2 pages:297-+ ispartof: location:England status: published

Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock., eLife digest Anyone who has crossed multiple time zones on a long flight will be familiar with jet lag, and that feeling of wanting to sleep at lunchtime and eat in the middle of the night. Many bodily processes, including appetite and wakefulness, roughly follow a 24-hour cycle. These cycles are known as circadian rhythms, from the Latin ‘circa diem’ meaning about a day. An area of the brain called the suprachiasmatic nucleus (SCN) coordinates circadian rhythms. It acts as a master clock by generating a 24-hour signal for the rest of the body to follow. Jet lag occurs when this internal circadian rhythm becomes out of sync with the local day-night cycle. Although jet lag can be uncomfortable, it tends to disappear over the course of a few days. This is because exposure to daylight in our new location resets the SCN master clock, enabling us to adapt to a new time zone. But evidence suggests that long-term disruption of circadian rhythms, for example as a result of shift work, may have lasting harmful effects. These include an increased risk of degenerative brain disorders such as Parkinson's disease and Alzheimer's disease. Brenna et al. now identify a molecular mechanism that could explain this link. A key component of the SCN master clock is a protein called Period2 (PER2). Levels of PER2 rise and fall over each 24-hour period, helping the brain keep track of time. Brenna et al. show that PER2 interacts with CDK5, a protein that helps regulate brain development and that has been implicated in Parkinson's disease and Alzheimer's disease. Reducing CDK5 levels in mice shortened their circadian rhythms by several hours. It also altered the animals’ behavioral patterns over a 24-hour period. Deleting the gene for PER2 had a similar effect, suggesting that CDK5 helps regulate PER2. Future studies should investigate the molecular links between CDK5, circadian rhythms and processes such as neurodegeneration. The results would provide clues to whether manipulating the circadian clock could help prevent or treat neurological disorders.

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.

Macroautophagy/autophagy, a highly conserved dynamic process, is one of the major degradative pathways in cells. So far, over 40 autophagy-related (ATG) genes have been identified in Saccharomyces cerevisiae, most of which have homologs in more complex eukaryotes. Autophagy plays a crucial role in cell survival and maintenance, and its dysfunction is related to various diseases, indicating that the proper regulation of autophagy is important. Although the overall process of autophagy has been extensively studied, in particular with regard to the function of the Atg proteins, relatively little is known about the regulatory mechanisms that control autophagy activity. Spt5 is one of the transcriptional factors that is universally conserved across all domains. This protein can form a complex with Spt4, together playing a central role in transcription. In complex eukaryotic cells, the Spt4-Spt5 complex plays a dual role in gene regulation, acting both to delay transcription through promoter-proximal pausing, and to facilitate transcriptional elongation. In contrast, in S. cerevisiae, only the positive function of the Spt4-Spt5 complex has been identified. Here, we show for the first time that the Spt4-Spt5 transcription factor complex negatively regulates ATG genes in S. cerevisiae, inhibiting autophagy activity during active growth. Under autophagy-inducing conditions, the repression is released by Spt5 phosphorylation, allowing an upregulation of autophagy activity. AID: auxin-inducible degron; ATG: autophagy-related; ChIP: chromatin immunoprecipitation;Cvt: cytoplasm-to-vacuole targeting; DSIF: DRB sensitivity-inducible factor; NELF: negativeelongation factor; ORF: open reading frame; PA: protein A; PE: phosphatidylethanolamine;prApe1: precursor aminopeptidase I; RT-qPCR: real-time quantitative PCR; RNAP II: RNApolymerase II; TSS: transcription start site; WT: wild-type

The nucleotide-binding domain- and leucine-rich repeat (LRR)-containing proteins (NLRs) function as intracellular immune receptors to detect the presence of pathogen- or host-derived signals. The mechanisms of how NLRs sense their ligands remain elusive. Here we report the structure of a bacterial flagellin derivative in complex with the NLR proteins NAIP5 and NLRC4 determined by cryo-electron microscopy at 4.28 A resolution. The structure revealed that the flagellin derivative forms two parallel helices interacting with multiple domains including BIR1 and LRR of NAIP5. Binding to NAIP5 results in a nearly complete burial of the flagellin derivative, thus stabilizing the active conformation of NAIP5. The extreme C-terminal side of the flagellin is anchored to a sterically constrained binding pocket of NAIP5, which likely acts as a structural determinant for discrimination of different bacterial flagellins by NAIP5, a notion further supported by biochemical data. Taken together, our results shed light on the molecular mechanisms underlying NLR ligand perception.

Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down ofCdk5in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Therefore, CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by the circadian clock.

Macroautophagy/autophagy, a highly conserved dynamic process, is one of the major degradative pathways in cells. So far, over 40 autophagy-related (ATG) genes have been identified in Saccharomyces cerevisiae, most of which have homologs in more complex eukaryotes. Autophagy plays a crucial role in cell survival and maintenance, and its dysfunction is related to various diseases, indicating that the proper regulation of autophagy is important. Although the overall process of autophagy has been extensively studied, in particular with regard to the function of the Atg proteins, relatively little is known about the regulatory mechanisms that control autophagy activity. Spt5 is one of the transcriptional factors that is universally conserved across all domains. This protein can form a complex with Spt4, together playing a central role in transcription. In complex eukaryotic cells, the Spt4-Spt5 complex plays a dual role in gene regulation, acting both to delay transcription through promoter-proximal pausing, and to facilitate transcriptional elongation. In contrast, in S. cerevisiae, only the positive function of the Spt4-Spt5 complex has been identified. Here, we show for the first time that the Spt4-Spt5 transcription factor complex negatively regulates ATG genes in S. cerevisiae, inhibiting autophagy activity during active growth. Under autophagy-inducing conditions, the repression is released by Spt5 phosphorylation, allowing an upregulation of autophagy activity.