Intracellular signaling pathways are modulated by ubiquitin-dependent trafficking, in which specific plasma membrane receptors and cytosolic proteins are tagged, internalized, and degraded in the endolysosomal pathway. Target of Myb1 (TOM1) family proteins, including TOM1, TOM1-L1, and TOM1-L2 function as early adaptors within the ESCRT-0 machinery to recognize ubiquitinated cargo and coordinate its sorting. TOM1 proteins interact with ubiquitin and accessory proteins, such as TOLLIP, facilitating efficient cargo sequestration and endosomal maturation. These interactions are known to be modulated by pathogen-driven processes, such as Shigella flexneri-mediated phosphatidylinositol 5-phosphate accumulation, which can impair TOM1-dependent cargo trafficking. Beyond endosomal sorting, TOM1 contributes to autophagic flux by linking autophagosomes and endosomes through its interaction with the motor protein Myosin VI. Dysregulation of these pathways has been implicated in immune disorders, myocardial ischemia-reperfusion injury, and potentially tumorigenesis. In plants, TOM1-like proteins serve as functional ESCRT-0 analogs, mediating ubiquitin-dependent cargo sorting and integrating stress-responsive signaling. Recent studies have shed light on the modular organization of TOM1, revealing mechanisms of ubiquitin recognition, DXXLL motif function, and complex formation with adaptor proteins. Nonetheless, key questions remain regarding how TOM1 discriminates among ubiquitin linkages, interacts with distinct phosphoinositides under varying physiological conditions, and cooperates with TOLLIP during selective autophagy. Elucidating these mechanisms will advance our understanding of cellular transport and signaling and may reveal novel intervention targets for inflammatory and autoimmune diseases in humans as well as for improving drought tolerance and immune regulation in plants.

Cholesteryl esters (CEs) and triacylglycerols (TAGs) are stored in lipid droplets (LDs), but their compartmentalisation is not well understood. Here, we established a hyperspectral stimulated Raman scattering microscopy system to identify and quantitatively assess CEs and TAGs in individual LDs of human cells. We found that nuclear envelope-associated lipid droplets (NE-LDs) were enriched in cholesteryl esters compared to lipid droplets in the cytoplasm. Correlative light-volume-electron microscopy revealed that NE-LDs projected towards the cytoplasm and associated with type II nuclear envelope (NE) invaginations. The nuclear envelope localization of sterol O-acyltransferase 1 (SOAT1) contributed to NE-LD generation, as trapping of SOAT1 to the NE further increased their number. Upon stimulation by the pro-inflammatory cytokine TNFα, the number of NE-LDs moderately increased. Moreover, TNFα-induced NF-κB nuclear translocation was fine-tuned by SOAT1: increased SOAT1 activity and NE-LDs associated with faster NF-κB translocation, whereas reduced SOAT1 activity and NE-LDs associated with slower NF-κB translocation. Our findings suggest that the NE is enriched in CEs and that cholesterol esterification can modulate nuclear translocation.

A recently described G307D variant of the endosomal adaptor protein TOM1 causes severe early-onset multiorgan autoimmunity and combined immunodeficiency. By combining biophysical, biochemical and cell culture experiments, we show that the variant causes a defect in the interaction between TOM1 and TOLLIP, another adaptor protein involved in cargo trafficking and regulation of innate immunity. The G307D variant impairs the ability of TOM1 to reduce TOLLIP phosphatidylinositol 3-phosphate binding, an important regulatory mechanism for cargo trafficking commitment for both proteins. Our experiments using TOM1 G307D patient cells suggested that the variant affects autophagy, seen as an aggravated response to amino acid starvation and accumulation of autophagosomes due to autophagosome-lysosome fusion defect. In addition, inflammatory pathways showed excessive activation in TOM1 G307D patient cells. Our data suggest that the interaction between TOM1 and TOLLIP has a role in the regulation of the human immune system and highlight the importance of fundamental cellular functions, such as cargo trafficking, in controlling immune responses. Our study also provides insights into the caveats of immunomodulatory and stem cell therapies in patients with TOM1 pathogenic variants.

The microprotein adipogenin (Adig) is predominantly expressed in adipose tissues. Here, we found that Adig interacts with seipin to form a stable, rigid complex. We present the structure of the seipin-Adig complex at an overall resolution of ~3.0 angstroms. The structure revealed that mammalian seipin assembles into two distinct oligomeric forms: undecamers and dodecamers. Adig selectively bound to the dodecameric form and enhanced seipin assembly by bridging and stabilizing adjacent subunits. Functionally, this complex promoted lipid droplet development at both early and late stages. In transgenic mice, adipocyte-specific overexpression of Adig increased fat mass and enlarged lipid droplets, whereas Adig deletion disrupted triglyceride accumulation in brown adipose tissues. Thus, Adig can modulate lipid storage through its structural and functional interactions with seipin.

Sac1 is a conserved phosphoinositide phosphatase, whose loss-of-function compromises cell and organism viability. Here, we employ acute auxin-inducible Sac1 degradation to identify its immediate downstream effectors in human cells. Most of Sac1 is degraded in ~1 h, paralleled by increased PI(4)P and decreased cholesterol in the trans-Golgi network (TGN) during the following hour, and superseded by Golgi fragmentation, impaired glycosylation, and selective degradation of TGN proteins by ~4 h. The TGN disintegration results from its acute deacidification caused by disassembly of the Golgi V-ATPase. Mechanistically, Sac1 mediated TGN membrane composition maintains an assembly-promoting conformation of the V0a2 subunit. Key phenotypes of acute Sac1 degradation are recapitulated in human differentiated trophoblasts, causing processing defects of chorionic gonadotropin, in line with loss-of-function intolerance of the human SACM1L gene. Collectively, our findings reveal that the assembly of the Golgi V-ATPase is controlled by the TGN membrane via Sac1 fuelled lipid exchange.

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.

Hypercholesterolemia has long been implicated in endothelial cell (EC) dysfunction, but the mechanisms by which excess cholesterol causes vascular pathology are incompletely understood. Here we used a cholesterol-mimetic probe to map cholesterol-protein interactions in primary human ECs and discovered that cholesterol binds to and stabilizes the adhesion molecule VCAM-1. We show that accessible plasma membrane (PM) cholesterol in ECs is acutely responsive to inflammatory stimuli and that the nonvesicular cholesterol transporter Aster-A regulates VCAM-1 stability in activated ECs by controlling the size of this pool. Deletion of Aster-A in ECs increases VCAM-1 protein, promotes immune cell recruitment to vessels, and impairs pulmonary immune homeostasis. Conversely, depleting cholesterol from the endothelium in vivo dampens VCAM-1 induction in response to inflammatory stimuli. These findings identify cholesterol binding to VCAM-1 as a key step during EC activation and provide a biochemical explanation for the ability of excess membrane cholesterol to promote immune cell recruitment to the endothelium.

This review discusses how research around the oxysterol-binding protein family has evolved. We briefly summarize how this protein family, designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins, was discovered, how protein domains highly conserved among family members between taxa paved the way for understanding their mechanisms of action, and how insights into protein structural and functional features help to understand their versatility as lipid transporters. We also discuss questions and future avenues of research opened by these findings. The investigations on oxysterol-binding protein family serve as a real-life example of the notion that science often advances as a collective effort of multiple lines of enquiry, including serendipitous routes. While original articles invariably explain the motivation of the research undertaken in rational terms, the actual paths to findings may be less intentional. Fortunately, this does not reduce the impact of the discoveries made. Besides hopefully providing a useful account of ORP family proteins, we aim to convey this message.

Adipogenin (Adig) is an evolutionarily conserved microprotein and is highly expressed in adipose tissues and testis. Here, we identify Adig as a critical regulator for lipid droplet formation in adipocytes. We determine that Adig interacts directly with seipin, leading to the formation of a rigid complex. We solve the structure of the seipin/Adig complex by Cryo-EM at 2.98Å overall resolution. Surprisingly, seipin can form two unique oligomers, undecamers and dodecamers. Adig selectively binds to the dodecameric seipin complex. We further find that Adig promotes seipin assembly by stabilizing and bridging adjacent seipin subunits. Functionally, Adig plays a key role in generating lipid droplets in adipocytes. In mice, inducible overexpression of Adig in adipocytes substantially increases fat mass, with enlarged lipid droplets. It also elevates thermogenesis during cold exposure. In contrast, inducible adipocyte-specific Adig knockout mice manifest aberrant lipid droplet formation in brown adipose tissues and impaired cold tolerance.

Our study uncovers for the first time the role of TOM1-TOLLIP interaction in the regulation of the human immune system. A recently described G307D variant in the GAT domain of the endosomal adaptor protein TOM1 causes severe early-onset multiorgan autoimmunity and features of combined immunodeficiency. Through a combination of biophysical, biochemical, and cell culture experiments, we show that the variant causes a defect in the interaction of TOM1 and TOLLIP, another adaptor protein involved in cargo trafficking and regulation of innate immunity. The G307D variant deteriorates the ability of TOM1 to reduce TOLLIP’s phosphatidylinositol 3-phosphate binding, an important regulatory mechanism for cargo trafficking commitment for both proteins. TOM1 G307D patient cells demonstrated aberrant autophagy manifested as an aggravated response to amino acid starvation and an autophagosome-lysosome fusion defect leading to accumulation of autophagosomes. Key inflammatory pathways showed exaggerated activation in TOM1 G307D patient cells, providing a plausible explanation for the patients’ phenotype. Our data highlight the importance of fundamental cellular functions such as cargo trafficking in the regulation of the human immune system and provide insights into the caveats of immunomodulatory and stem cell therapies in patients with TOM1 pathogenic variants.

BACKGROUND:Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms. METHODS:Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control. RESULTS:We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo. CONCLUSIONS:These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.

BACKGROUND:Many members of the oxysterol-binding protein-related protein (ORP) family have been characterized in detail over the past decades, but the lipid transport and other functions of ORP7 still remain elusive. What is known about ORP7 points toward an endoplasmic reticulum and plasma membrane-localized protein, which also interacts with GABA type A receptor-associated protein like 2 (GABARAPL2) and unlipidated Microtubule-associated proteins 1A/1B light chain 3B (LC3B), suggesting a further autophagosomal/lysosomal association. Functional roles of ORP7 have been suggested in cholesterol efflux, hypercholesterolemia, and macroautophagy. We performed a hypothesis-free multi-omics analysis of chemical ORP7 inhibition utilizing transcriptomics and lipidomics as well as proximity biotinylation interactomics to characterize ORP7 functions in a primary cell type, human umbilical vein endothelial cells (HUVECs). Moreover, assays on angiogenesis, cholesterol efflux, and lipid droplet quantification were conducted. RESULTS:Pharmacological inhibition of ORP7 leads to an increase in gene expression related to lipid metabolism and inflammation, while genes associated with cell cycle and cell division were downregulated. Lipidomic analysis revealed increases in ceramides and lysophosphatidylcholines as well as saturated and monounsaturated triacylglycerols. Significant decreases were seen in all cholesteryl ester and in some unsaturated triacylglycerol species, compatible with the detected decrease of mean lipid droplet area. Along with the reduced lipid stores, ATP-binding cassette subfamily G member 1 (ABCG1)-mediated cholesterol efflux and angiogenesis decreased. Interactomics revealed an interaction of ORP7 with AKT1, a central metabolic regulator. CONCLUSIONS:The transcriptomics results suggest an increase in prostanoid as well as oxysterol synthesis, which could be related to the observed upregulation of proinflammatory genes. We envision that the defective angiogenesis in HUVECs subjected to ORP7 inhibition could be the result of an unfavorable plasma membrane lipid composition and/or reduced potential for cell division. To conclude, the present study suggests multifaceted functions of ORP7 in lipid homeostasis, angiogenic tube formation, and gene expression of lipid metabolism, inflammation, and cell cycle in primary endothelial cells.

Cholesterol is an essential lipid species of mammalian cells. Cells acquire it through synthesis in the endoplasmic reticulum (ER) and uptake from lipoprotein particles. Newly synthesized cholesterol is efficiently distributed from the ER to other organelles via lipid-binding/transfer proteins concentrated at membrane contact sites (MCSs) to reach the trans-Golgi network, endosomes, and plasma membrane. Lipoprotein-derived cholesterol is exported from the plasma membrane and endosomal compartments via a combination of vesicle/tubule-mediated membrane transport and transfer through MCSs. In this review, we provide an overview of intracellular cholesterol trafficking pathways, including cholesterol flux from the ER to other membranes, cholesterol uptake from lipoprotein donors and transport from the plasma membrane to the ER, cellular cholesterol efflux to lipoprotein acceptors, as well as lipoprotein cholesterol secretion from enterocytes, hepatocytes, and astrocytes. We also briefly discuss human diseases caused by defects in these processes and therapeutic strategies available in such conditions.

Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

Cellular cholesterol can be metabolized to its fatty acid esters, cholesteryl esters (CEs), to be stored in lipid droplets (LDs). With triacylglycerols (TGs), CEs represent the main neutral lipids in LDs. However, while TG melts at ~4 °C, CE melts at ~44 °C, raising the question of how CE-rich LDs form in cells. Here, we show that CE forms supercooled droplets when the CE concentration in LDs is above 20% to TG and, in particular, liquid-crystalline phases when the fraction of CEs is above 90% at 37 °C. In model bilayers, CEs condense and nucleate droplets when the CE/phospholipid ratio reaches over 10-15%. This concentration is reduced by TG pre-clusters in the membrane that thereby facilitate CE nucleation. Accordingly, blocking TG synthesis in cells is sufficient to strongly dampen CE LD nucleation. Finally, CE LDs emerged at seipins, which cluster and nucleate TG LDs in the ER. However, when TG synthesis is inhibited, similar numbers of LDs are generated in the presence and absence of seipin, suggesting that seipin controls CE LD formation via its TG clustering capacity. Our data point to a unique model whereby TG pre-clusters, favorable at seipins, catalyze the nucleation of CE LDs. Dumesnil et al. report that cholesterol esters (CE), which only melt above body temperature, form supercooled liquid crystalline lipid droplets (LD). Triacylglycerols (TG) solubilize CE to help CE LD nucleation. Through clustering TGs in the ER membrane, seipin controls CE LD nucleation sites.

Figure S1 shows cell growth and lipid distribution data. Figure S2 shows lipid distribution and invasion data. Figure S3 shows additional transcriptomic data. Figure S4 and S5 shows additional orthotopic data.

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that is activated by membrane binding and dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 has a minor effect on bulk autophagic flux, DFCP1 is required to maintain the autophagic flux of p62 under both fed and starved conditions, and this is dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localize to forming omegasomes, these omegasomes fail to constrict properly in a size-dependent manner. Consequently, the release of nascent autophagosomes from large omegasomes is markedly delayed. While knockout of DFCP1 does not affect bulk autophagy, it inhibits selective autophagy, including aggrephagy, mitophagy and micronucleophagy. We conclude that DFCP1 mediates ATPase-driven constriction of large omegasomes to release autophagosomes for selective autophagy.

Supplementary Data from Lipid Metabolic Reprogramming Extends beyond Histologic Tumor Demarcations in Operable Human Pancreatic Cancer

Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN‐independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1‐dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity. Whether removal of defective mitochondria is cause or consequence of metabolic reprogramming in highly energy‐demanding cells remains unclear. This study reports metabolic effects of mitochondrial iron depletion, as well as unexpected synergy between lipid droplet (LD) biogenesis and mitophagy sustaining cell and tissue integrity. DGAT1‐dependent lipid homeostasis synergizes with PINK1/Parkin‐independent mitophagy to sustain cell and tissue integrity.