Search

Introduction Deep learning approaches have shown high diagnostic performance in image classifications, such as differentiation of malignant tumors and calcified coronary plaque. However, it is unknown whether deep learning is useful for characterizing coronary plaques without the presence of calcification using coronary computed tomography angiography (CCTA). The purpose of this study was to compare the diagnostic performance of deep learning with a convolutional neural network (CNN) with that of radiologists in the estimation of coronary plaques. Methods We retrospectively enrolled 178 patients (191 coronary plaques) who had undergone CCTA and integrated backscatter intravascular ultrasonography (IB-IVUS) studies. IB-IVUS diagnosed 81 fibrous and 110 fatty or fibro-fatty plaques. We manually captured vascular short-axis images of the coronary plaques as Portable Network Graphics (PNG) images (150 × 150 pixels). The display window level and width were 100 and 700 Hounsfield units (HU), respectively. The deep-learning system (CNN; GoogleNet Inception v3) was trained on 153 plaques; its performance was tested on 38 plaques. The area under the curve (AUC) obtained by receiver operating characteristic analysis of the deep learning system and by two board-certified radiologists was compared. Results With the CNN, the AUC and the 95% confidence interval were 0.83 and 0.69–0.96, respectively; for radiologist 1 they were 0.61 and 0.42–0.80; for radiologist 2 they were 0.68 and 0.51–0.86, respectively. The AUC for CNN was significantly higher than for radiologists 1 (p = 0.04); for radiologist 2 it was not significantly different (p = 0.22). Conclusion DL-CNN performed comparably to radiologists for discrimination between fatty and fibro-fatty plaque on CCTA images. Implications for practice The diagnostic performance of the CNN and of two radiologists in the assessment of 191 ROIs on CT images of coronary plaques whose type corresponded with their IB-IVUS characterization was comparable.

SummaryUbiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Herein, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and subsequently CDK5RAP3, the last of which is an adaptor for ufmylating ribosomal subunit RPL26. When CDK5RAP3 was absent from the E3 complex, UFBP1 ufmylation occurred, a process thought to drive ER-phagy. Further, upon treatment with anisomycin, an inducer of disome formation, the UFM1 E3 complex associated with ufmylated RPL26 on the 60S ribosomal subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results clarify the molecular mechanism of the UFM1 system and provide new insights into the role of ufmylation.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of phosphorylation remain unclear. Herein, we identify ULK1 as a kinase responsible for phosphorylation of p62. ULK1 co-localizes with p62 bodies, and directly interacts with p62. This phosphorylation allows KEAP1 to be retained within p62 bodies, activating NRF2.p62S351E/+mice are phosphomimetic knock-in mice in which Ser351 corresponding to human Ser349 is replaced by Glu. These mice, but not phosphodefectivep62S351A/S351Amice, exhibit NRF2 hyperactivation and growth retardation, the latter caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis.p62S351E/+mice are a phenocopy of systemicKeap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insight into the role of phase separation in this process.

Autophagy is an intracellular degradation system regulating cellular homeostasis. The two ubiquitin-like modification systems named the Atg8 system and the Atg12 system are essential for autophagy. Atg8 and Atg12 are ubiquitin-like proteins covalently conjugated with a phosphatidylethanolamine (PE) and Atg5, respectively, via enzymatic reactions. The Atg8-PE conjugate binds to autophagic membranes and recruits various proteins through direct interaction, whereas the Atg12-Atg5 conjugate recognizes Atg3, the E2 enzyme for Atg8, and facilitates Atg8-PE conjugation by functioning as the E3 enzyme. Although structural and biochemical analyses have well established the Atg8-family interacting motif (AIM), studies on the interacting sequence for Atg12 are rare (only one example for human ATG12-ATG3), thereby making it challenging to define a binding motif. Here we determined the crystal structure of the plant ATG12b as a complex with the ATG12b-binding region of ATG3 and revealed that ATG12b recognizes the aspartic acid (Asp)-methionine (Met) motif in ATG3 via a hydrophobic pocket and a basic residue, which we confirmed critical for the complex formation by mutational analysis. This recognition mode is similar to that reported between human ATG12 and ATG3, suggesting that the Asp-Met sequence is a conserved Atg12-interacting motif (AIM12). These data suggest that AIM12 mediates E2-E3 interaction during Atg8 lipidation and provide structural basis for developing chemicals that regulate autophagy by targeting Atg12-family proteins.

Selective modulation of autophagy is a promising therapeutic strategy, especially for cancer treatment. However, the lack of specific autophagy inhibitors limits this strategy. The formation of the ATG12-ATG5-ATG16L1 complex is essential for targeting the ATG12-ATG5 conjugate to proper membranes and to generate LC3-II for the progression of autophagy. Thus, targeting ATG5-ATG16L1 protein-protein interactions (PPIs) might inhibit early stage autophagy with high specificity. In this paper, we report that a stapled peptide derived from ATG16L1 exhibits potent binding affinity to ATG5, striking resistance to proteolysis, and significant autophagy inhibition activities in cells.

Introduction With intra-arterial digital subtraction angiography (DSA) considered as the gold standard, we compared the diagnostic value of computed tomography angiography (CTA) and computed tomography-digital subtraction angiography (CT-DSA in hemodialysis (HD) patients suspected of having lower limb peripheral artery disease (PAD). Methods In this retrospective study, we enrolled 220 HD patients with suspected PAD. CT-DSA images were obtained by subtracting unenhanced images from enhanced images. The research team calculated the area under the curve (AUC), sensitivity, specificity, positive and negative predictive value (PPV, NPV), and recorded the diagnostic accuracy between the CTA and CT-DSA images using the DSA as gold standard. Visual evaluation of calcifications in the peripheral arteries were also compared between CTA and CT-DSA images. Results At the above-knee level, the CTA AUC [95% confidence interval (CI)] was 0.68 (CI 0.64–0.72), sensitivity and specificity were 60 and 81%, PPV and NPV were 85 and 53%, and accuracy was 67%. Below the knee, these values were 0.66 (CI 0.62–0.70), 71 and 79%, 79 and 47%, and 66%. For CT-DSA, above-knee, the AUC [95% CI] was 0.88 (CI 0.85–0.91), sensitivity and specificity were 84 and 92%, PPV and NPV were 89 and 97%, and accuracy was 93%. Below the knee, these values were 0.95 (CI 0.93–0.97), 95 and 93%, 96 and 83%, and 93%. The scores for the visualization of calcification in the peripheral arteries was significantly higher for CT-DSA than CTA (p Conclusions CT-DSA helps to assess stenotic PAD with high calcification in the lower extremities of HD patients. Implications for practice On CT-DSA images, the severity of vascular calcification can be assessed for HD patients suspected of PAD of the lower extremities.

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis. Lipidated Atg8 affects membrane morphology via two aromatic membrane-facing residues that are important for autophagy in budding yeast and mammalian cells.

Selective autophagy cargos are recruited to autophagosomes primarily by interacting with autophagosomal ATG8 family proteins via the LC3‐interacting region (LIR). The upstream sequence of most LIRs contains negatively charged residues such as Asp, Glu, and phosphorylated Ser and Thr. However, the significance of LIR phosphorylation (compared with having acidic amino acids) and the structural basis of phosphorylated LIR–ATG8 binding are not entirely understood. Here, we show that the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)‐phagy receptor TEX264 are phosphorylated by casein kinase 2, which is critical for its interaction with ATG8s, autophagosomal localization, and ER‐phagy. Structural analysis shows that phosphorylation of these serine residues increases binding affinity by producing multiple hydrogen bonds with ATG8s that cannot be mimicked by acidic residues. This binding mode is different from those of other ER‐phagy receptors that utilize a downstream helix, which is absent from TEX264, to increase affinity. These results suggest that phosphorylation of the LIR is critically important for strong LIR–ATG8 interactions, even in the absence of auxiliary interactions.

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles. The ER is subject to autophagy (ER-phagy) for turnover, with Atg40 acting as a receptor to sequester ER with Atg8 in autophagosomes. Here, the authors show that Atg40 is clustered by interaction with Atg8 to generate local membrane curvature and promote autophagosome packing.

Many biomolecules undergo liquid–liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes—double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3–5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein–protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS. The pre-autophagosomal structure in yeast is a liquid-like condensate of Atg proteins whose phase separation may have a critical, active role in autophagy.

Selective autophagy cargos are recruited to autophagosomes primarily by interacting with autophagosomal ATG8 family proteins via the LC3-interacting region (LIR). The upstream sequence of most LIRs contains negatively charged residues such as Asp, Glu, and phosphorylated Ser and Thr. However, the significance of LIR phosphorylation (compared with having acidic amino acids) and the structural basis of phosphorylated LIR–ATG8 binding are not entirely understood. Here, we show that the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)-phagy receptor TEX264 are phosphorylated by casein kinase 2, which is critical for its interaction with ATG8s, autophagosomal localization, and ER-phagy. Structural analysis showed that phosphorylation of these serine residues increased binding affinity by producing multiple hydrogen bonds with ATG8s that cannot be mimicked by acidic residues. This binding mode is different from those of other ER-phagy receptors that utilize a downstream helix, which is absent from TEX264, to increase affinity. These results suggest that phosphorylation of the LIR is critically important for strong LIR–ATG8 interactions, even in the absence of auxiliary interactions.

Mammals conserve multiple mammalian Atg8-family proteins (mATG8s) consisting of GABARAP (GABA type A receptor-associated protein) and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) subfamilies that tightly bind to autophagic membranes in a membrane-anchored form. These proteins are crucial for selective autophagy and recruit proteins bearing LC3-interacting region (LIR) motifs. However, because limited research tools are available, information on the specific roles of each membrane-anchored mATG8 in selective autophagy is scarce. In this study, we identified LIR motifs specific to the membrane-anchored form of each mATG8 and characterized the residues critical for their selective interaction using cell-based assays and structural analyses. We then used these selective LIR motifs to develop probes and irreversible deconjugases that targeted selective membrane-anchored mATG8s in the autophagic membrane, revealing that membrane-anchored GABARAP subfamily proteins regulate the aggrephagy of amyotrophic lateral sclerosis-linked protein aggregates. Our tools will be useful for elucidating the functional significance of each mATG8 protein on autophagic membranes in autophagy research. A:C autophagic membrane:cytosol; ALS amyotrophic lateral sclerosis; ATG4 autophagy related 4; Atg8 autophagy related 8; BafA1 bafilomycin A1; BNIP3L/Nix BCL2 interacting protein 3 like; CALCOCO2/NDP52 calcium binding and coiled-coil domain 2; EBSS Earle’s balanced salt solution; GABARAP GABA type A receptor-associated protein; GST glutathione S transferase; HKO hexa knockout; Kd dissociation constant; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NLS nuclear localization signal/sequence; PE phosphatidylethanolamine; SpHfl1 Schizosaccharomyces pombeorganic solute transmembrane transporter; SQSTM1/p62 SQSTM1/p62; TARDBP/TDP-43 TAR DNA binding protein; TKO triple knockout

TOR complex 1 (TORC1) is an evolutionarily-conserved protein kinase that controls cell growth and metabolism in response to nutrients, particularly amino acids. In mammals, several amino acid sensors have been identified that converge on the multi-layered machinery regulating Rag GTPases to trigger TORC1 activation; however, these sensors are not conserved in many other organisms including yeast. Previously, we reported that glutamine activates yeast TORC1 via a Gtr (Rag ortholog)-independent mechanism involving the vacuolar protein Pib2, although the identity of the supposed glutamine sensor and the exact TORC1 activation mechanism remain unclear. In this study, we successfully reconstituted glutamine-responsive TORC1 activation in vitro using only purified Pib2 and TORC1. In addition, we found that glutamine specifically induced a change in the folding state of Pib2. These findings indicate that Pib2 is a glutamine sensor that directly activates TORC1, providing a new model for the metabolic control of cells., Tanigawa et al. investigate the role of Pib2, a vacuolar protein, in the glutamine-dependent upstream regulation of TORC1 in budding yeast. Their study reveals that Pib2 plays dual roles as a glutamine sensor and a direct TORC1 activator, a finding useful for the community working on TORC1 upstream regulation.

Atg8 has attracted attention as a central factor in autophagosome biogenesis for a long time. However, the molecular activities of Atg8 on the phagophore membranes as the physiologically functional lipidated form remain enigmatic. In our recent study, we unveiled the hidden physicochemical activity of lipidated Atg8 toward the membrane. Structural analysis revealed that lipidated Atg8 adopts a preferred orientation on the membrane, contacting the membrane using aromatic residues and at the same time exposing cargo binding pockets to the solvent, enabling this small protein to perturb and transform membranes while recognizing autophagic cargos. The membrane perturbation activity was shown to be essential for efficient autophagosome biogenesis, yet questions on the mechanistic roles of Atg8 remain open.

Summary Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid-liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget’s disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the anti-oxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein-protein interaction assay, doxycycline-inducible gene expression system and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.

SummaryA series of amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43 and hnRNPA1 has an ability to be liquid-liquid phase separation, and their disease-related mutations cause the transition of their responsible liquid droplets to aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD) and Paget’s disease of bone. SQSTM1/p62 protein forms liquid-droplets through the interaction with ubiquitinated proteins, and the droplet serves as a platform of autophagosome formation and anti-oxidative stress response via the LC3-interacting region (LIR) and Keap1-interacting region (KIR), respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in LIR and KIR form aberrant liquid droplets, cause defective autophagy and stress response or both. To evaluate the effects of ALS/FTD-related p62 mutations in LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway and the autophagic turnover, we developed systems that enable to monitor them with high sensitivity. These systems revealed that some mutants but not all have their less abilities on the Nrf2-activation and show the delayed turnover. By contrast, while the sufficient ability to form liquid droplets, all droplets consisting of p62 mutants showed slower inner fluidity. These results indicate that like other ALS-related mutant proteins, a primary defect in ALS/FTD with p62 missense mutations is a qualitative change of p62-liquid droplets.

The degradation of cytoplasmic components via autophagy is crucial for intracellular homeostasis. In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy‐related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate de novo organelle biogenesis.

Breeding of blue flowers in commercially important ornamental plants, such as chrysanthemum and rose, is relatively difficult by conventional breeding techniques because there are no closely-related species with blue flowers. Therefore, the creation of novel blue flowers has been attempted in various ornamental plants by using biotechnology. As a result, bluish colored carnations and roses have been produced and successfully commercialized. Our research group has also succeeded in developing technology based on genetic engineering to confer the pure blue flower color trait to chrysanthemum (Chrysanthemum morifolium). The blue coloration of the chrysanthemum was accomplished by constructing functional genes producing flavonoid 3’,5’-hydroxylase and anthocyanin 3’,5’-O-glucosyltransferase. Accumulation of 3’,5’-O-glucosylated delphinidin-based anthocyanins synthesized by the transgenes led to the development of blue color via intermolecular copigmentation with flavone glycosides present in chrysanthemum petal. Subsequently, by introducing the two genes necessary for blue petal coloration, we succeeded in conferring the blue flower color trait to various chrysanthemums with different floral shapes and color shades. Successful commercialization of blue chrysanthemums should facilitate an expansion of flower color variation, increase potential flower value, and raise new possibilities and applications for chrysanthemums. Furthermore, in vitro experiments suggested that, in addition to flavone glycosides, flavonol glycosides might have an effect on 3’,5’-O-glucosylated delphinidin-based anthocyanins as blue-coloring copigments in planta. These results indicate that the method established in chrysanthemums for the blue color development of petals by hydroxylation and glucosylation of the B-ring of anthocyanins is likely to be applicable to other ornamental plants.

Ubiquitin or ubiquitin-like proteins can be covalently conjugated to multiple proteins that do not necessarily have binding interfaces. Here, we show that an evolutionary transition from covalent conjugation to non-covalent interaction has occurred in the ubiquitin-like autophagy-related 12 (ATG12) conjugation system. ATG12 is covalently conjugated to its sole substrate, ATG5, by a ubiquitylation-like mechanism. However, the apicomplexan parasites Plasmodium and Toxoplasma and some yeast species such as Komagataella phaffii (previously Pichia pastoris) lack the E2-like enzyme ATG10 and the most carboxy (C)-terminal glycine of ATG12, both of which are required for covalent linkage. Instead, ATG12 in these organisms forms a non-covalent complex with ATG5. This non-covalent ATG12-ATG5 complex retains the ability to facilitate ATG8-phosphatidylethanolamine conjugation. These results suggest that ubiquitin-like covalent conjugation can evolve to a simpler non-covalent interaction, most probably when the system has a limited number of targets.

Autophagy is an intracellular degradation system conserved among eukaryotes that mediates the degradation of various biomolecules and organelles. During autophagy, a double membrane-bound organelle termed an autophagosome is synthesized de novo and delivers targets from the cytoplasm to the lysosomes for degradation. Autophagosome formation involves complex and dynamic membrane rearrangements, which are regulated by dozens of autophagy-related (Atg) proteins. In this review, we summarize our current knowledge of membrane-binding domains and motifs in Atg proteins and discuss their roles in autophagy.

Autophagy is an intracellular degradation system involving de novo generation of autophagosomes, which function as a transporting vesicle of cytoplasmic components to lysosomes for degradation. Isolation membranes (IMs) or phagophores, the precursor membranes of autophagosomes, require millions of phospholipids to expand and transform into autophagosomes, with the endoplasmic reticulum (ER) being the primary lipid source. Recent advances in structural and biochemical studies of autophagy-related proteins have revealed their lipid transport activities: Atg2 at the interface between IM and ER possesses intermembrane lipid transfer activities, while Atg9 at IM and VMP1 and TMEM41B at ER possess lipid scrambling activities. In this review, we summarize recent advances in the establishment of the lipid transport activities of these proteins and their collaboration mechanisms for lipid transport between the ER and IM, and further discuss how unidirectional lipid transport from the ER to IM occurs during autophagosome formation.

Point-mutations of MEK1, a central component of ERK signaling, are present in cancer and RASopathies, but their precise biological effects remain obscure. Here, we report a mutant MEK1 structure that uncovers the mechanisms underlying abnormal activities of cancer- and RASopathy-associated MEK1 mutants. These two classes of MEK1 mutations differentially impact on spatiotemporal dynamics of ERK signaling, cellular transcriptional programs, gene expression profiles, and consequent biological outcomes. By making use of such distinct characteristics of the MEK1 mutants, we identified cancer- and RASopathy-signature genes that may serve as diagnostic markers or therapeutic targets for these diseases. In particular, two AKT-inhibitor molecules, PHLDA1 and 2, are simultaneously upregulated by oncogenic ERK signaling, and mediate cancer-specific ERK-AKT crosstalk. The combined expression of PHLDA1/2 is critical to confer resistance to ERK pathway-targeted therapeutics on cancer cells. Finally, we propose a therapeutic strategy to overcome this drug resistance. Our data provide vital insights into the etiology, diagnosis, and therapeutic strategy of cancers and RASopathies.

Mammals conserve multiple mammalian ATG8 proteins (mATG8s) consisting of γ-aminobutyric acid receptor-associated protein (GABARAP) and microtubule-associated protein 1 light-chain 3 (LC3) subfamilies that tightly bind to the autophagic membranes in a lipidated form. They are crucial in selective autophagy and recruit proteins bearing LC3-interacting region (LIR) motifs. However, because limited research tools are available, information about the specific roles of each lipidated mATG8 in selective autophagy is scarce. Here, we identified LIR motifs specific to the lipidated form of each mATG8 and characterized the residues critical for their selective interaction using cell-based assays and structural analyses. Then, we used these selective LIR motifs to develop probes and irreversible deconjugases that targeted selective lipidated mATG8s in the autophagic membrane, revealing that lipidated GABARAP subfamily proteins regulate aggrephagy of amyotrophic lateral sclerosis-linked protein aggregates. Our tools will be useful in elucidating the functional significance of each mATG8 protein in autophagy research.

Autophagy, an intracellular degradation mechanism, involves de novo generation of autophagosomes that sequester and deliver cytoplasmic components to the lysosome for degradation. The mechanism behind autophagosomal membrane expansion has been a longstanding enigma in this field. Recent structural and biochemical analyses have revealed that two mysterious autophagy-related (Atg) proteins, Atg2 and Atg9, are novel types of intermembrane and interleaflet lipid transporters, respectively. This review summarizes recent discoveries surrounding Atg2 and Atg9 as a lipid transporter and discusses the molecular mechanism of autophagosomal membrane expansion driven by collaboration between these two lipid transporters.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions and promotes several pathological events, including anaphylaxis and neuropathic pain. PAF is biosynthesized by two types of lyso-PAF acetyltransferases: lysophosphatidylcholine acyltransferase 1 (LPCAT1) and LPCAT2, which are constitutive and inducible forms of lyso-PAF acetyltransferase, respectively. Because LPCAT2 mainly produces PAF under inflammatory stimuli, understanding the structure of LPCAT2 is important for developing specific drugs against PAF-related inflammatory diseases. Although the structure of LPCAT2 has not been determined, the crystal structure was reported for Thermotoga maritima PlsC, an enzyme in the same gene family as LPCAT2. Here, we identified residues in mouse LPCAT2 essential for its enzymatic activity and a potential acyl-coenzyme A (CoA)-binding pocket, based on homology modeling of mouse LPCAT2 with PlsC. We also found that Ala115 of mouse LPCAT2 was important for acyl-CoA selectivity. In conclusion, these results predict the three-dimensional (3D) structure of mouse LPCAT2. Our findings have implications for the future development of new drugs against PAF-related diseases.

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress., Liquid-liquid phase separation of p62/SQSTM1 has been previously described, although the significance in vivo remains unclear. Here the authors show p62 droplets contain ubiquitin, autophagy-related proteins and Keap1 to serve as platform of not only autophagosome formation but also Nrf2 activation.

International audience; High-speed AFM imaging enables a semiquantitative, realistic description of the dynamic structure of intrinsically disordered proteins.Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

Autophagy, which is an evolutionarily conserved intracellular degradation system, involves de novo generation of autophagosomes that sequester and deliver diverse cytoplasmic materials to the lysosome for degradation. Autophagosome formation is mediated by approximately 20 core autophagy-related (Atg) proteins, which collaborate to mediate complicated membrane dynamics during autophagy. To elucidate the molecular functions of these Atg proteins in autophagosome formation, many researchers have tried to determine the structures of Atg proteins by using various structural biological methods. Although not sufficient, the basic structural catalog of all core Atg proteins was established. In this review article, we summarize structural biological studies of core Atg proteins, with an emphasis on recently unveiled structures, and describe the mechanistic breakthroughs in autophagy research that have derived from new structural information.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions and promotes several pathological conditions, including anaphylaxis and neuropathic pain. PAF is biosynthesized by two types of lyso-PAF acetyltransferases: lysophosphatidylcholine acyltransferase 1 (LPCAT1) and LPCAT2, which are constitutive and inducible forms of lyso-PAF acetyltransferase, respectively. Because LPCAT2 mainly produces PAF under inflammatory conditions, understanding the structure of LPCAT2 is important for developing specific drugs against PAF-related inflammatory diseases. Although the structure of LPCAT2 has not been determined, the crystal structure was reported forThermotoga maritimaPlsC, an enzyme in the same enzyme family as LPCAT2. In this study, we identified residues in mouse LPCAT2 essential for its enzymatic activity and a potential acyl-coenzyme A (CoA)-binding pocket, based on homology modeling of mouse LPCAT2 with PlsC. We also found that Ala115 of mouse LPCAT2 was important for acyl-CoA selectivity. In conclusion, these results predict the structure of mouse LPCAT2. Our findings have implications for the future development of new drugs against PAF-related diseases.