Discovery of Small-Molecule Autophagy Inhibitors by Disrupting the Protein-Protein Interactions Involving Autophagy-Related 5.

One possible strategy for modulating autophagy is to disrupt the critical protein-protein interactions (PPIs) formed during this process. Our attention is on the autophagy-related 12 (ATG12)-autophagy-related 5 (ATG5)-autophagy-related 16-like 1 (ATG16L1) heterotrimer complex, which is responsible for ATG8 translocation from ATG3 to phosphatidylethanolamine. In this work, we discovered a compound with an (E)-3-(2-furanylmethylene)-2-pyrrolidinone core moiety (T1742) that blocked the ATG5-ATG16L1 and ATG5-TECAIR interactions in the in vitro binding assay (IC50 = 1-2 µM) and also exhibited autophagy inhibition in cellular assays. The possible binding mode of T1742 to ATG5 was predicted through molecular modeling, and a batch of derivatives sharing essentially the same core moiety were synthesized and tested. The outcomes of the in vitro binding assay and the flow cytometry assay of those newly synthesized compounds were generally consistent. This work has validated our central hypothesis that small-molecule inhibitors of the PPIs involving ATG5 can tune down autophagy effectively, and their pharmaceutical potential may be further explored.

The effect of lactoferrin on ULK1 and ATG13 genes expression in breast cancer cell line MCF7 and bioinformatics studies of protein interaction between lactoferrin and the autophagy initiation complex.

Recently, the study of autophagy and its mechanism on the cancer cell growth process has received much attention. lactoferrin (Lf) is a glycoprotein with various biological activities, including antibacterial, antiviral, anti-cancer, etc. In the present study, the effect of different concentrations of lactoferrin on the expression of ULK1 and ATG13 genes was evaluated in breast cancer cell line MCF7 using real-time PCR technique as well as the molecular mechanism of these two genes and their proteins in the autophagy pathway and the relationship between lactoferrin and these proteins were investigated by bioinformatics studies. The result showed that the expression of the ULK1 gene at a concentration of 500 µg/ml of lactoferrin was significantly (P < 0.007) increased compared to the control and two other concentrations. Also, the expression of the ATG13 gene at all three concentrations was not significantly different from each other and compared to the control (P = 0.635). In the immunoblot of ULK1 protein at a concentration of 500 µg, more protein expression was observed. The binding mode of lactoferrin with ULK1, ATG13, and ATG101 proteins was obtained using docking. According to docking results, the N-lobe region of lactoferrin interacts with the PS domain of the ULK1 protein, and the N-lobe region of lactoferrin interacts with the horma domain of the ATG 13 and ATG101 proteins. The results show that lactoferrin, in addition to acting on the gene, interacts with ULK1, ATG13, and ATG101 proteins. Since all three proteins are components of the autophagy initiation complex, lactoferrin can induce autophagy in this way.

Discovery and Structure-Based Optimization of Novel Atg4B Inhibitors for the Treatment of Castration-Resistant Prostate Cancer.

Autophagy inhibition is an attractive target for cancer therapy. In this study, we discovered inhibitors of Atg4B essential for autophagosome formation and evaluated their potential as therapeutics for prostate cancer. Seventeen compounds were identified as candidates after in silico screening and a thermal shift assay. Among them, compound 17 showed the most potent Atg4B inhibitory activity, inhibited autophagy induced by anti-castration-resistant prostate cancer (CRPC) drugs, and significantly enhanced apoptosis. Although 17 has been known as a phospholipase A2 (PLA2) inhibitor, other PLA2 inhibitors had no effect on Atg4B and autophagy. We then performed structural optimization based on molecular modeling and succeeded in developing 21f (by shortening the alkyl chain of 17), which was a potent competitive inhibitor for Atg4B (Ki = 3.1 µM) with declining PLA2 inhibitory potency. Compound 21f enhanced the anticancer activity of anti-CRPC drugs via autophagy inhibition. These findings suggest that 21f can be used as an adjuvant drug for therapy with anti-CRPC drugs.

Cycloastragenol alleviates airway inflammation in asthmatic mice by inhibiting autophagy.

Cycloastragenol (CAG), a secondary metabolite from the roots of Astragalus zahlbruckneri, has been reported to exert anti­inflammatory effects in heart, skin and liver diseases. However, its role in asthma remains unclear. The present study aimed to investigate the effect of CAG on airway inflammation in an ovalbumin (OVA)­induced mouse asthma model. The current study evaluated the lung function and levels of inflammation and autophagy via measurement of airway hyperresponsiveness (AHR), lung histology examination, inflammatory cytokine measurement and western blotting, amongst other techniques. The results demonstrated that CAG attenuated OVA­induced AHR in vivo. In addition, the total number of leukocytes and eosinophils, as well as the secretion of inflammatory cytokines, including interleukin (IL)­5, IL­13 and immunoglobulin E were diminished in bronchoalveolar lavage fluid of the OVA­induced murine asthma model. Histological analysis revealed that CAG suppressed inflammatory cell infiltration and goblet cell secretion. Notably, based on molecular docking simulation, CAG was demonstrated to bind to the active site of autophagy­related gene 4­microtubule­associated proteins light chain 3 complex, which explains the reduced autophagic flux in asthma caused by CAG. The expression levels of proteins associated with autophagy pathways were inhibited following treatment with CAG. Taken together, the results of the present study suggest that CAG exerts an anti­inflammatory effect in asthma, and its role may be associated with the inhibition of autophagy in lung cells.

Pathogenic variants of ATG4D in infertile men with non-obstructive azoospermia identified using whole-exome sequencing.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility, and it is primarily associated with genetic defects. We performed whole-exome sequencing of 236 patients with NOA and identified a homozygous pathogenic variant of autophagy-related 4D cysteine peptidase (ATG4D) in two siblings from a consanguineous family and compound heterozygous pathogenic variants of ATG4D in two sporadic cases. The expression of LC3B, a regulator of autophagic activity, was significantly decreased, and the apoptosis rate of spermatogenic cells in testicular tissues was increased. Transfection of GC-2spd cells with a ATG4D mutant plasmid (Flag-Atg4dmut ) significantly decreased the expression level of Lc3b and increased the rate of apoptosis. Moreover, a pathogenic variant in X-linked ATG4A and compound heterozygous pathogenic variants of ATG4B were identified in one patient each. All novel variants were segregated by disease phenotype and were predicted to be pathogenic. Our findings revealed that autophagy-related cysteine peptidase family genes may play crucial roles in human spermatogenesis and identified ATG4D as a novel candidate gene for male infertility due to NOA.

Persulfidation of ATG18a regulates autophagy under ER stress in Arabidopsis.

Hydrogen sulfide (H2S) is an endogenously generated gaseous signaling molecule, which recently has been implicated in autophagy regulation in both plants and mammals through persulfidation of specific targets. Persulfidation has been suggested as the molecular mechanism through which sulfide regulates autophagy in plant cells. ATG18a is a core autophagy component that is required for bulk autophagy and also for reticulophagy during endoplasmic reticulum (ER) stress. In this research, we revealed the role of sulfide in plant ER stress responses as a negative regulator of autophagy. We demonstrate that sulfide regulates ATG18a phospholipid-binding activity by reversible persulfidation at Cys103, and that this modification activates ATG18a binding capacity to specific phospholipids in a reversible manner. Our findings strongly suggest that persulfidation of ATG18a at C103 regulates autophagy under ER stress, and that the impairment of persulfidation affects both the number and size of autophagosomes.

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast.

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12-Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12-Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113-131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64-99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.

Identification of a novel differentially methylated region adjacent to ATG16L2 in lung cancer cells using methyl-CpG binding domain protein-enriched genome sequencing.

Lung cancer is the most common cancer worldwide. Epigenetic modifications like DNA methylation play fundamental roles in the dynamic process of lung cancer. The objective of this study was to use methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) to identify novel and high-confidence DNA methylation in lung tumor. We first compared the whole-genome DNA methylation of three lung cancer cell lines, including A549, H1299, and SK-MES-1, against BEAS-2B, a lung/bronchial normal epithelial cell line. We then used pyrosequencing and OneStep qMethyl kit methods to verify the results in the cell line specimens. MBD-Seq identified 279, 8046, and 22 887 differentially methylated regions (DMRs), respectively, with 120 common DMRs among three comparison groups. Three DMRs were consistent with the MBD-Seq results by both pyrosequencing and OneStep qMethyl validations. Furthermore, OneStep qMethyl kit was also performed for functional validation of these three potential DMRs in sputum DNA from clinical participants. We successfully identified one new DMR adjacent to ATG16L2. The novel DMR might have an important function in lung carcinogenesis. Further validation of the finding in clinical specimens of lung cancer patients and functional analysis of this novel DMR in the development of lung cancer through transcriptional silencing of ATG16L2 are warranted.

Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery.

Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin-proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.

Structure of the C9orf72 ARF GAP complex that is haploinsufficient in ALS and FTD.

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.

ULK1 and ULK2 are less redundant than previously thought: computational analysis uncovers distinct regulation and functions of these autophagy induction proteins.

Macroautophagy, the degradation of cytoplasmic content by lysosomal fusion, is an evolutionary conserved process promoting homeostasis and intracellular defence. Macroautophagy is initiated primarily by a complex containing ULK1 or ULK2 (two paralogs of the yeast Atg1 protein). To understand the differences between ULK1 and ULK2, we compared the human ULK1 and ULK2 proteins and their regulation. Despite the similarity in their enzymatic domain, we found that ULK1 and ULK2 have major differences in their autophagy-related interactors and their post-translational and transcriptional regulators. We identified 18 ULK1-specific and 7 ULK2-specific protein motifs serving as different interaction interfaces. We found that interactors of ULK1 and ULK2 all have different tissue-specific expressions partially contributing to diverse and ULK-specific interaction networks in various tissues. We identified three ULK1-specific and one ULK2-specific transcription factor binding sites, and eight sites shared by the regulatory region of both genes. Importantly, we found that both their post-translational and transcriptional regulators are involved in distinct biological processes-suggesting separate functions for ULK1 and ULK2. Unravelling differences between ULK1 and ULK2 could lead to a better understanding of how ULK-type specific dysregulation affects autophagy and other cellular processes that have been implicated in diseases such as inflammatory bowel disease and cancer.

Distinct Mechanisms for Processing Autophagy Protein LC3-PE by RavZ and ATG4B.

Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens in eukaryotic cells. Lipidated LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. Endogenous ATG4-mediated deconjugation of LC3-PE is required for LC3 recycling. However, the Legionella effector RavZ irreversibly deconjugates LC3-PE to inhibit autophagy. It is not clear how ATG4 and RavZ process LC3-PE with distinct modes. Herein, a series of semisynthetic LC3-PE proteins containing C-terminal mutations or insertions were used to investigate the relationship of the C-terminal structure of LC3-PE with ATG4/RavZ-mediated deconjugation. Using a combination of molecular docking and biochemical assays, we found that Gln116, Phe119 and Gly120 of LC3-PE are required for cleavage by both RavZ and ATG4B, whereas Glu117(LC3) is specific to cleavage by RavZ. The molecular ruler mechanism exists in the active site of ATG4B, but not in RavZ. Met63 and Gln64 at the active site of RavZ are involved in accommodating LC3 C-terminal motif. Our findings show that the distinct binding modes of the LC3 C-terminal motif (116-120) with ATG4 and RavZ might determine the specificity of cleavage site.

Multi-site-mediated entwining of the linear WIR-motif around WIPI ß-propellers for autophagy.

WIPI proteins (WIPI1-4) are mammalian PROPPIN family phosphoinositide effectors essential for autophagosome biogenesis. In addition to phosphoinositides, WIPI proteins can recognize a linear WIPI-interacting-region (WIR)-motif, but the underlying mechanism is poorly understood. Here, we determine the structure of WIPI3 in complex with the WIR-peptide from ATG2A. Unexpectedly, the WIR-peptide entwines around the WIPI3 seven-bladed ß-propeller and binds to three sites in blades 1-3. The N-terminal part of the WIR-peptide forms a short strand that augments the periphery of blade 2, the middle segment anchors into an inter-blade hydrophobic pocket between blades 2-3, and the C-terminal aromatic tail wedges into another tailored pocket between blades 1-2. Mutations in three peptide-binding sites disrupt the interactions between WIPI3/4 and ATG2A and impair the ATG2A-mediated autophagic process. Thus, WIPI proteins recognize the WIR-motif by multi-sites in multi-blades and this multi-site-mediated peptide-recognition mechanism could be applicable to other PROPPIN proteins.

Unstructured Biology of Proteins from Ubiquitin-Proteasome System: Roles in Cancer and Neurodegenerative Diseases.

The 26S proteasome is a large (~2.5 MDa) protein complex consisting of at least 33 different subunits and many other components, which form the ubiquitin proteasomal system (UPS), an ATP-dependent protein degradation system in the cell. UPS serves as an essential component of the cellular protein surveillance machinery, and its dysfunction leads to cancer, neurodegenerative and immunological disorders. Importantly, the functions and regulations of proteins are governed by the combination of ordered regions, intrinsically disordered protein regions (IDPRs) and molecular recognition features (MoRFs). The structure-function relationships of UPS components have not been identified completely; therefore, in this study, we have carried out the functional intrinsic disorder and MoRF analysis for potential neurodegenerative disease and anti-cancer targets of this pathway. Our report represents the presence of significant intrinsic disorder and disorder-based binding regions in several UPS proteins, such as extraproteasomal polyubiquitin receptors (UBQLN1 and UBQLN2), proteasome-associated polyubiquitin receptors (ADRM1 and PSMD4), deubiquitinating enzymes (DUBs) (ATXN3 and USP14), and ubiquitinating enzymes (E2 (UBE2R2) and E3 (STUB1) enzyme). We believe this study will have implications for the conformation-specific roles of different regions of these proteins. This will lead to a better understanding of the molecular basis of UPS-associated diseases.

Molecular cloning, expression and functional analysis of Atg16L1 from orange-spotted grouper (Epinephelus coioides).

Autophagy related gene 16 (Atg16), which encodes a core protein for autophagosome formation, participates in autophagy activity, the ubiquitin proteasome system and inflammatory response in mammals. In this study, we cloned and characterized an Atg16 homolog from orange-spotted grouper (Epinephelus coioides) (EcAtg16L1). EcAtg16L1 encodes a 656-amino acid polypeptide, which shares 94.22% and 72.65% homology with large yellow croakers (Larimichthys crocea) and humans (Homo sapiens), respectively. EcAtg16L1 contains a conserved Atg16 domain and a WD-repeat-containing domain. Subcellular localization showed that EcAtg16L1 was distributed in the cytoplasm of grouper cells with a dot-like pattern. EcAtg16L1 overexpression promoted Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by the increase in viral gene transcription and viral coat protein. Furthermore, EcAtg16L1 overexpression negatively regulated interferon (IFN)-related molecules and proinflammatory cytokines, and decreased IFN, IFN-stimulated response element, and nuclear factor κB promoter activities. Taken together, aside from its function in autophagosome formation, EcAtg16L1 also plays role in promoting SGIV and RGNNV replication and the pro-viral effect might involve its down regulation to interferon and inflammatory responses.

Design, Synthesis, Antibacterial Potential, and Structural Characterization of N-Acylated Derivatives of the Human Autophagy 16 Polypeptide.

A synthetic antimicrobial peptide library based on the human autophagy 16 polypeptide has been developed. Designed acetylated peptides bearing lipids of different chain lengths resulted in peptides with enhanced potency compared to the parent Atg16. A 21-residue fragment of Atg16 conjugated to 4-methylhexanoic acid (K30) emerged as the most potent antibacterial, with negligible hemolysis. Several studies, including microscopy, dye leakage, and ITC, were conducted to gain insight into the antibacterial mechanism of action of the peptide. Visual inspection using both SEM and TEM revealed the membranolytic effect of the peptide on bacterial cells. The selectivity of the peptide against bacterial cell membranes was also proven using dye leakage assays. ITC analysis revealed the exothermic nature of the binding interaction of the peptide to D8PG micelles. The three-dimensional solution NMR structure of K30 in complex with dioctanoylphosphatidylglycerol (D8PG) micelles revealed that the peptide adopts a helix-loop-helix structure in the presence of anionic membrane lipids mimicking bacterial membranes. Intermolecular NOEs between the peptide and lipid deciphered the location of the peptide in the bound state, which was subsequently supported by the paramagnetic relaxation enhancement (PRE) NMR experiment. Collectively, these results describe the structure-function relationship of the peptide in the bacterial membrane.

ATG10 (autophagy-related 10) regulates the formation of autophagosome in the anti-virus immune response of pacific oyster (Crassostrea gigas).

Autophagy, a highly conserved intracellular degradation system, is involved in numerous processes in vertebrate and invertebrate, such as cell survival, ageing, and immune responses. However, the detailed molecular mechanism of autophagy and its immune regulatory role in bivalves are still not well understood. In the present study, an autophagy-related protein ATG10 (designated as CgATG10) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgATG10 cDNA was of 621 bp, encoding a polypeptide of 206 amino acid residues with an Autophagy_act_C domain (from 96 to 123 amino acid), which shared high homology with that from C. virginica and Octopus bimaculoides. The mRNA transcripts of CgATG10 were widely expressed in all the tested tissues including mantle, gonad, gills, hemocytes and hepatopancreas, with the highest expression level in mantle. After the stimulation with poly (I:C), the mRNA expression level of CgATG10 in the mantle of oysters was significantly up-regulated (4.92-fold of that in Blank group, p < 0.05), and the LC3-conversion from LC3-I to LC3-II (LC3-II/LC3-I) also increased. After an additional injection of dsRNA to knock-down the expression of CgATG10 (0.33-fold and 0.10-fold compared respectively with Blank group and dsGFP group, p < 0.05), the downstream conversion of CgLC3 was inhibited significantly compared with that of the control dsGFP group, while the expression level of autophagy-initiator CgBeclin1 did not change significantly. In addition, the mRNA transcripts of interferon regulatory factor CgIRF-1 increased significantly in CgATG10-knockdown oysters at 12 h post poly (I:C) stimulation. All the results indicated that CgATG10 might participate in the immune response against poly (I:C) by regulating autophagosome formation and interferon system in oysters.

Intrinsically Disordered Protein TEX264 Mediates ER-phagy.

Certain proteins and organelles can be selectively degraded by autophagy. Typical substrates and receptors of selective autophagy have LC3-interacting regions (LIRs) that bind to autophagosomal LC3 and GABARAP family proteins. Here, we performed a differential interactome screen using wild-type LC3B and a LIR recognition-deficient mutant and identified TEX264 as a receptor for autophagic degradation of the endoplasmic reticulum (ER-phagy). TEX264 is an ER protein with a single transmembrane domain and a LIR motif. TEX264 interacts with LC3 and GABARAP family proteins more efficiently and is expressed more ubiquitously than previously known ER-phagy receptors. ER-phagy is profoundly blocked by deletion of TEX264 alone and almost completely by additional deletion of FAM134B and CCPG1. A long intrinsically disordered region of TEX264 is required for its ER-phagy receptor function to bridge the gap between the ER and autophagosomal membranes independently of its amino acid sequence. These results suggest that TEX264 is a major ER-phagy receptor.

The epg5 knockout zebrafish line: a model to study Vici syndrome.

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Structural Conservation of the Two Phosphoinositide-Binding Sites in WIPI Proteins.

WIPI proteins are mammalian PROPPIN family members that bind to phosphoinositides and play prominent roles in autophagosome biogenesis. Two phosphoinositide-binding sites were previously described in yeast PROPPIN Hsv2 but remain to be determined in mammalian WIPI proteins. Here, we characterized four human WIPI proteins (WIPI1-4) and solved the structure of WIPI3. WIPI proteins can bind to PI(3)P and PI(3,5)P2 and adopt a conventional seven-bladed ß-propeller fold. The structure of WIPI3 revealed that WIPI proteins also contain two sites embedded in blades 5 and 6 for recognizing phosphoinositides, resembling that in Hsv2. Structural comparison further demonstrated that the two conserved phosphoinositide-binding sites in PROPPIN proteins are not identical but intrinsically tend to recognize different types of phosphoinositides. This work provides the structural evidence to support the conservation of the two phosphoinositide-binding sites in WIPI proteins and also uncovers the potential phosphoinositide-binding selectivity for each site.