Downregulation of Lnc-ABCA12-3 modulates UBQLN1 expression and protein homeostasis pathways in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration. Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in ALS pathogenesis but their roles remain unclear. Previous studies found lnc-ABCA12-3 was downregulated in ALS patients. We aim to characterize the expression and function of lnc-ABCA12-3 in ALS and explore its mechanisms of action. Lnc-ABCA12-3 expression was analyzed in PBMCs from ALS patients and correlated with clinical outcomes. Effect of modulating lnc-ABCA12-3 expression was assessed in cell models using assays of apoptosis, protein homeostasis and pathway analysis. RNA pull-down and interaction studies were performed to identify lnc-ABCA12-3 binding partners. Lnc-ABCA12-3 was downregulated in ALS patients, correlating with faster progression and shorter survival. Overexpression of lnc-ABAC12-3 conferred protection against oxidative stress-induced apoptosis, while knockdown lnc-ABCA12-3 enhanced cell death. Lnc-ABCA12-3 maintained protein quality control pathways, including ubiquitination, autophagy and stress granule formation, by regulating the ubiquitin shuttle protein UBQLN1. This study identified lnc-ABCA12-3 as a novel regulatory lncRNA implicated in ALS pathogenesis by modulating cellular survival and stress responses through interactions with UBQLN1, influencing disease progression. Lnc-ABCA12-3 may influence ALS through regulating protein homeostasis pathways.

Structure-activity relationship study of small-molecule inhibitor of Atg12-Atg3 protein-protein interaction.

Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation. In our study, we identified compound 1 as an inhibitor of the ATG12-ATG3 protein-protein interaction. We focused on the systematic modification of the original hit 1, a casein kinase 2 (CK2) inhibitor, to find potent disruptors of ATG12-ATG3 protein-protein interaction. A systematic modification of the hit structure led us to a wide plethora of compounds that maintain its ATG12-ATG3 inhibitory activity, which could act as a viable starting point to design new compounds with diverse therapeutic applications.

The role of autophagy in RIP1 mediated cell death and intestinal inflammation.

Autophagy, a highly conserved catabolic process that targets various types of cellular cargoes to lysosomal degradation, is one of the most important biological mechanisms critical for cellular homeostasis. Components of these cellular cargoes can range from individual proteins to invading pathogens, and degrading these materials is important for maintaining organismal health and survival. The process of autophagy is carried out by complex molecular mechanisms, and a growing body of evidence indicates that these mechanisms intersect with those involved in the cell death pathways. In this review, we examine several emerging studies elucidating the role of autophagy in RIP1-mediated cell death signaling, with particular emphasis on impaired autophagy caused by ATG16L1 deficiency. We also discuss how autophagy in RIP1-mediated cell death affects intestinal homeostasis in preclinical models, and the implications of the intersection between RIP1 and autophagy for understanding the intestinal pathologies associated with inflammatory bowel disease (IBD). Finally, we highlight the potential benefits of therapeutic targeting of RIP1 and autophagy proteins, while also proposing areas of research that will likely elucidate new links between autophagy and cell death signaling.

Absence of ATG9A and synaptophysin demixing on Rab5 mutation-induced giant endosomes.

ATG9A is the only integral membrane protein among core autophagy-related (ATG) proteins. We previously found that ATG9A does not co-assemble into synaptophysin-positive vesicles, but rather, localizes to a distinct pool of vesicles within synapsin condensates in both fibroblasts and nerve terminals. The endocytic origin of these vesicles further suggests the existence of different intracellular sorting or segregation mechanisms for ATG9A and synaptophysin in cells. However, the precise underlying mechanism remains largely unknown. In this follow-up study, we investigated the endosomal localization of these two proteins by exploiting the advantages of a Rab5 mutant that induces the formation of enlarged endosomes. Notably, ATG9A and synaptophysin intermix perfectly and do not segregate on giant endosomes, indicating that the separation of these two proteins is not solely caused by the inherent properties of the proteins, but possibly by other unknown factors.

Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication.

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus-host interactions and potential antiviral strategies.

Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit.

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate.

Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.

Sulfur starvation-induced autophagy in Saccharomyces cerevisiae involves SAM-dependent signaling and transcription activator Met4.

Autophagy is a key lysosomal degradative mechanism allowing a prosurvival response to stresses, especially nutrient starvation. Here we investigate the mechanism of autophagy induction in response to sulfur starvation in Saccharomyces cerevisiae. We found that sulfur deprivation leads to rapid and widespread transcriptional induction of autophagy-related (ATG) genes in ways not seen under nitrogen starvation. This distinctive response depends mainly on the transcription activator of sulfur metabolism Met4. Consistently, Met4 is essential for autophagy under sulfur starvation. Depletion of either cysteine, methionine or SAM induces autophagy flux. However, only SAM depletion can trigger strong transcriptional induction of ATG genes and a fully functional autophagic response. Furthermore, combined inactivation of Met4 and Atg1 causes a dramatic decrease in cell survival under sulfur starvation, highlighting the interplay between sulfur metabolism and autophagy to maintain cell viability. Thus, we describe a pathway of sulfur starvation-induced autophagy depending on Met4 and involving SAM as signaling sulfur metabolite.

Role of ATG4 Autophagy-Related Protein Family in the Lower Airways of Patients with Stable COPD.

Autophagy is a complex physiological pathway mediating homeostasis and survival of cells degrading damaged organelles and regulating their recycling. Physiologic autophagy can maintain normal lung function, decrease lung cellular senescence, and inhibit myofibroblast differentiation. It is well known that autophagy is activated in several chronic inflammatory diseases; however, its role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and the expression of autophagy-related genes (ATGs) in lower airways of COPD patients is still controversial. The expression and localization of all ATG proteins that represented key components of the autophagic machinery modulating elongation, closure, and maturation of autophagosome membranes were retrospectively measured in peripheral lungs of patients with stable COPD (n = 10), control smokers with normal lung function (n = 10), and control nonsmoking subjects (n = 8) using immunohistochemical analysis. These results show an increased expression of ATG4 protein in alveolar septa and bronchiolar epithelium of stable COPD patients compared to smokers with normal lung function and non-smoker subjects. In particular, the genes in the ATG4 protein family (including ATG4A, ATG4B, ATG4C, and ATG4D) that have a key role in the modulation of the physiological autophagic machinery are the most important ATGs increased in the compartment of lower airways of stable COPD patients, suggesting that the alteration shown in COPD patients can be also correlated to impaired modulation of autophagic machinery modulating elongation, closure, and maturation of autophagosomes membranes. Statistical analysis was performed by the Kruskal-Wallis test and the Mann-Whitney U test for comparison between groups. A statistically significant increased expression of ATG4A (p = 0.0047), ATG4D (p = 0.018), and ATG5 (p = 0.019) was documented in the bronchiolar epithelium as well in alveolar lining for ATG4A (p = 0.0036), ATG4B (p = 0.0054), ATG4C (p = 0.0064), ATG4D (p = 0.0084), ATG5 (p = 0.0088), and ATG7 (p = 0.018) in patients with stable COPD compared to control groups. The ATG4 isoforms may be considered as additional potential targets for the development of new drugs in COPD.

Estrogen receptor is involved in the osteoarthritis mediated by Atg16L1-NLRP3 activation.

OBJECTIVES: This study aims to explore the mechanisms of dual regulation of osteoarthritis (OA) progression by the involvement of estrogen receptor (ER) in autophagy and inflammation. MATERIALS AND METHODS: Bioinformatics methods were used to explore the relationship among associated genes. Western blot assays were used to detect related protein expression of OA in C28I2 and induced OA cellular model. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis were used to detect OA related gene expression in C28I2 and induced OA cellular model. Co-immunoprecipitation (CO-IP) analysis were used to verify the direct interaction between ER and NOD-like receptor thermal protein domain associated protein 3 (NLRP3). RESULTS: The C28I2 cellular model of OA was induced by interleukin-1ß (IL-1ß). The small interfering ribonucleic acid (SiRNA)-mediated knockdown of autophagy-related 16 like 1 (ATG16L1) in C28I2 decreased the expression of MAP1LC3B (LC3B) and NLRP3. Besides, ER-beta (ERß) agonist changed the gene expression of NLRP3 and ATG16L1. Moreover, CO-IP analysis indicated the direct interaction between ER and NLRP3. CONCLUSION: Our study results revealed that ATG16L1, NLRP3, and IL-1ß interacted closely and ERß was involved in OA process by affecting autophagy and inflammatory activation.

Conjugation of ATG8s to single membranes at a glance.

Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.

WIPI2b recruitment to phagophores and ATG16L1 binding are regulated by ULK1 phosphorylation.

One of the key events in autophagy is the formation of a double-membrane phagophore, and many regulatory mechanisms underpinning this remain under investigation. WIPI2b is among the first proteins to be recruited to the phagophore and is essential for stimulating autophagy flux by recruiting the ATG12-ATG5-ATG16L1 complex, driving LC3 and GABARAP lipidation. Here, we set out to investigate how WIPI2b function is regulated by phosphorylation. We studied two phosphorylation sites on WIPI2b, S68 and S284. Phosphorylation at these sites plays distinct roles, regulating WIPI2b's association with ATG16L1 and the phagophore, respectively. We confirm WIPI2b is a novel ULK1 substrate, validated by the detection of endogenous phosphorylation at S284. Notably, S284 is situated within an 18-amino acid stretch, which, when in contact with liposomes, forms an amphipathic helix. Phosphorylation at S284 disrupts the formation of the amphipathic helix, hindering the association of WIPI2b with membranes and autophagosome formation. Understanding these intricacies in the regulatory mechanisms governing WIPI2b's association with its interacting partners and membranes, holds the potential to shed light on these complex processes, integral to phagophore biogenesis.

Unbiased MD simulations identify lipid binding sites in lipid transfer proteins.

The characterization of lipid binding to lipid transfer proteins (LTPs) is fundamental to understand their molecular mechanism. However, several structures of LTPs, and notably those proposed to act as bridges between membranes, do not provide the precise location of their endogenous lipid ligands. To address this limitation, computational approaches are a powerful alternative methodology, but they are often limited by the high flexibility of lipid substrates. Here, we develop a protocol based on unbiased coarse-grain molecular dynamics simulations in which lipids placed away from the protein can spontaneously bind to LTPs. This approach accurately determines binding pockets in LTPs and provides a working hypothesis for the lipid entry pathway. We apply this approach to characterize lipid binding to bridge LTPs of the Vps13-Atg2 family, for which the lipid localization inside the protein is currently unknown. Overall, our work paves the way to determine binding pockets and entry pathways for several LTPs in an inexpensive, fast, and accurate manner.

Docosahexaenoic acid enhances the treatment efficacy for castration-resistant prostate cancer by inhibiting autophagy through Atg4B inhibition.

Autophagy induction in cancer is involved in cancer progression and the acquisition of resistance to anticancer agents. Therefore, autophagy is considered a potential therapeutic target in cancer therapy. In this study, we found that long-chain fatty acids (LCFAs) have inhibitory effects on Atg4B, which is essential for autophagosome formation, through screening based on the pharmacophore of 21f, a recently developed Atg4B inhibitor. Among these fatty acids, docosahexaenoic acid (DHA), a polyunsaturated fatty acid, exhibited the most potent Atg4B inhibitory activity. DHA inhibited autophagy induced by androgen receptor signaling inhibitors (ARSI) in LNCaP and 22Rv1 prostate cancer cells and significantly increased apoptotic cell death. Furthermore, we investigated the effect of DHA on resistance to ARSI by establishing darolutamide-resistant prostate cancer 22Rv1 (22Rv1/Dar) cells, which had developed resistance to darolutamide, a novel ARSI. At baseline, 22Rv1/Dar cells showed a higher autophagy level than parental 22Rv1 cells. DHA significantly suppressed Dar-induced autophagy and sensitized 22Rv1/Dar cells by inducing apoptotic cell death through mitochondrial dysfunction. These results suggest that DHA supplementation may improve prostate cancer therapy with ARSI.

ATG10-dependent autophagy is required for DDX10 to regulate cell proliferation, apoptosis and stemness in colorectal cancer.

Colorectal cancer (CRC) remains a highly prevalent gastrointestinal neoplasm, presenting significant prevalence and lethality rate. DEAD/H box RNA helicase 10 (DDX10) has been proposed as a potential oncogene in CRC, the specific action mechanism by which DDX10 modulates the aggressive biological cellular events in CRC remains implicitly elucidated, however. During this study, DDX10 expression was detected via RT-qPCR and Western blotting. Cell proliferation was estimated via EDU staining. TUNEL staining and Western blotting appraised cell apoptosis. Cell stemness was evaluated by sphere formation assay, RT-qPCR, Western blotting as well as immunofluorescence staining. Relevant assay kit examined aldehyde dehydrogenase (ALDH) activity. Western blotting and immunofluorescence staining also detected autophagy. DDX10 was hyper-expressed in CRC cells. Down-regulation of DDX10 hampered cell proliferation, aggravated the apoptosis while eliminated the ability to form spheroid cells in CRC. In addition, DDX10 deletion improved ATG10 expression and therefore activated autophagy in CRC cells. Consequently, ATG10 depletion or treatment with autophagy inhibitor 3-Methyladenine (3-MA) partially compensated the influences of DDX10 silencing on the proliferation, apoptosis and stemness of CRC cells. Accordingly, DDX10 deficiency may aggravate autophagy mediated by ATG10 to impede cell proliferation, stemness and facilitate cell apoptosis, hence blocking the progression of CRC.

Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.

Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.

Determination of the Cryo-EM Structure of ATG9 from Arabidopsis thaliana.

Macroautophagy/autophagy is a highly conserved process for the degradation of cellular components and plays an essential role in cellular homeostasis maintenance. During autophagy, specialized double-membrane vesicles known as autophagosomes are formed and sequester cytoplasmic cargoes and deliver them to lysosomes or vacuoles for breakdown. Central to this process are autophagy-related (ATG) proteins, with the ATG9-the only integral membrane protein in this core machinery-playing a central role in mediating autophagosome formation. Recent years have witnessed the maturation of cryo-electron microscopy (cryo-EM) and single-particle analysis into powerful tools for high-resolution structural determination of protein complexes. These advancements have significantly deepened our understanding of the intricate molecular mechanisms underlying autophagosome biogenesis. In this study, we present a protocol detailing the acquisition of the three-dimensional structure of ATG9 from Arabidopsis thaliana. The structural resolution achieved 7.8 Å determined by single-particle cryo-electron microscopy (cryo-EM).

The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.