Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

The neutrophil NADPH oxidase is activated by signals generated by formyl peptide receptor (FPR) agonists recognized by FPR1 (fMLF) and FPR2 (WKYMVM), respectively. Also, the antagonists cyclosporin H (specific for FPR1) and PBP10 (specific for FPR2) inhibit the NADPH oxidase activity when induced by the two peptide agonists. When bound to its receptor, the non-activating positive allosteric modulator Cmp58, specific for the free fatty acid 2 receptor (FFA2R), affects not only the response induced by agonists specific for FFA2R, but also the activating potency but not the efficacy of the two FPR activating peptides. Even though Cmp58 is without effect on the efficacy of the response induced by the FPR agonists, the inhibitory effect of the respective FPR specific antagonists is reduced. This sensitivity shift was reversed by an FFA2R specific antagonist suggesting that two different signals generated by the FPRs activate the NADPH oxidase. According to a receptor trans-regulation model, the activated FPRs generate signals that directly activate the NADPH oxidase and signals that activate the allosterically modulated FFA2Rs to elicit activation of the NADPH.

Ubp3 is one of about 20 deubiquitinases (DUBs) in S. cerevisiae. The current view generally assumes that Ubp3 requires its interaction partner Bre5, which is proposed to function as a positive regulator. Accordingly, the Ubp3/Bre5 complex has been implicated in a broad range of cellular processes for example trafficking between ER and Golgi, stress granule formation and selective autophagy. However, the molecular basis of this proposed Bre5-dependent activity remains unclear. To address this at a molecular level, Ubp3, Bre5, and related constructs were heterologously expressed in E. coli, purified to homogeneity, and characterized in vitro. Both proteins contain folded domains as well as extensive intrinsically disordered regions (IDRs). Despite this structural complexity, the Ubp3/Bre5 complex could be isolated following either co-expression in vivo or after in vitro assembly. Unexpectedly, complex formation with Bre5 was not required for the catalytic activity of full length Ubp3. Furthermore, even the isolated catalytic domain of Ubp3 was fully active against two distinct substrates in the absence of Bre5, demonstrating that its deubiquitinating activity is intrinsically independent of Bre5. These findings indicate that the catalytic domain alone is sufficient for substrate cleavage, whereas the extensive IDRs of Ubp3 and its cofactor Bre5 might contribute to substrate recognition or specificity. Overall, this study challenges the prevailing model of Bre5-dependent activation of Ubp3 and provides new insights into the molecular organization of the Ubp3/Bre5 system. More broadly, it highlights the importance of intrinsically disordered regions in regulating deubiquitinase function and cellular signaling networks.

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

Phosphatidic acid (PA) phosphatase (PAP) is an enzyme that plays a major role in lipid signaling by controlling the cellular levels of two lipid secondary messengers: its substrate, PA, and its product, diacylglycerol. Two types of mammalian PAPs have been reported to date. Type 1 PAP (PAP1) is an Mg2+-dependent, N-ethylmaleimide (NEM)-sensitive cytosolic enzyme (EC 3.1.3.4), whereas type 2 PAP (PAP2), also known as phospholipid phosphate (PLPP) (EC 3.1.3.113), is an Mg2+-independent, NEM-insensitive transmembrane protein. PAP2 also hydrolyzes other bioactive lipids such as lyso-PA (LPA), sphingosine-1-phosphate (S1P), and ceramide-1-phosphate (C1P). Here, we purified human phosphatase orphan 2 (PHOSPHO2), a putative cytosolic phosphatase containing a haloacid dehalogenase-like domain, and characterized its enzymological properties in vitro. Purified PHOSPHO2 displays Mg2+-dependent, NEM-sensitive phosphatase activities toward PA, LPA, S1P, C1P, and glycerol-3-phosphate (G3P) in vitro. Moreover, PHOSPHO2 showed substrate selectivity for PA molecular species containing shorter saturated fatty acids such as lauric acid and myristic acid, or polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid. The PAP activity of PHOSPHO2, but not its other phosphatase activities, was strongly enhanced in the presence of phosphatidylcholine and phosphatidylethanolamine, major components of the cell membranes. These results indicate that mammalian PHOSPHO2 is a novel cytosolic PLPP that primarily functions as a PAP on cytoplasm-facing membranes.

The synthesis of mitochondrial-encoded polypeptides is an essential process, primarily regulated at the posttranscriptional level. In yeast, many regulatory factors have been described as acting in proximity to the mitoribosome to promote efficient translation; however, the precise mechanisms by which these components function remain largely unknown. Here, we expand on findings concerning a previously studied mitoribosome interactor, Mrx9, which is found in large expressosome-like assemblies of mitoribosome clusters. Mrx9 was initially linked to mitochondrial translation and was suggested to be associated with the splicing of COX1 and COB transcripts. Our current data show that Mrx9 is associated with the PHB/m-AAA complex at the polypeptide exit tunnel of the mitoribosome. Overexpression of Mrx9 impairs the proteolytic functions of Yta10 and Yta12 within the prohibitin complex, leading to splicing defects; accumulation of aberrant polypeptides; and a noticeable impairment in the processing of the essential mitoribosomal protein bL32m. These findings support a regulatory role for Mrx9 in the PHB/m-AAA complex by modulating the activities of both Yta10 and Yta12.

Extracellular nucleotides regulate diverse inflammatory responses through the activation of P2 receptors. Here, we show that nucleotides regulate TLR3-induced chemokine responses in murine bone marrow-derived macrophages (BMDMs). Stimulation with the TLR3 agonist, poly(I:C), induced CCL2 and CXCL1 secretion, whereas degradation of extracellular nucleotides with apyrase reduced CCL2 but enhanced CXCL1 release. In agreement, the non-selective P2 receptor antagonists suramin and RB2 reduced poly(I:C)-induced CCL2 production. Among the highly expressed P2 receptors examined, selective inhibition of P2Y6 with MRS2578 reproduced the effects of apyrase, decreasing CCL2 while increasing CXCL1 secretion. The absence of effects of MRS2578 in P2ry6-deficient BMDMs confirmed the specificity of this antagonist. Transcriptional analysis further revealed that P2Y6 inhibition reduced early poly(I:C)-induced Ccl2 and Cxcl10 expression while enhancing later Cxcl1 and Cxcl2 expression. In line with our previous works on other pattern-recognition receptors pathways and innate immune cells, this study further supports a role for extracellular nucleotides in shaping TLR-induced chemokine responses in macrophages.

Uncoupling protein 1 (UCP1) mediates thermogenesis in brown adipose tissue. Whether human-UCP1 shares the bioenergetic properties established for rodent UCP1 (innate uncoupling, GDP sensitivity, fatty acid (re)activation) is not known. Therefore, we expressed human and mouse UCP1 in mouse liver, using adeno-associated viral vectors, and characterized their properties in isolated liver mitochondria. Both UCP1s induced marked innate uncoupling, characterized by increased substrate-supported respiration and decreased membrane potential, in the absence of exogenous fatty acids. Mouse-UCP1 in liver retained the classical regulatory properties of native brown-fat UCP1, including potent inhibition by GDP and reactivation by oleate. In contrast, human-UCP1 was only weakly inhibited by GDP but was strongly responsive to fatty acids. However, ATP potently inhibited human-UCP1, with an apparent IC of {approx}0.4 mM compared with {approx}1.4 mM for GDP, and ATP markedly decreased the sensitivity of human-UCP1 to oleate (re)activation. Despite substantial UCP1-mediated uncoupling, oxidative phosphorylation capacity and mitochondrial OXPHOS protein levels were preserved. Molecular dynamics simulations suggested a structural basis for the species difference. GDP formed persistent interactions with F88 in mouse-UCP1, an interaction absent at the corresponding S88 residue in human-UCP1. In-silico substitution of F88 by serine reduced GDP interaction at this site. Thus, human and mouse UCP1 share innate thermogenic activity but differ fundamentally in nucleotide regulation. The F88/S88 difference may contribute to the preferential GDP sensitivity of mouse-UCP1, whereas ATP provides effective nucleotide control of human-UCP1.

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

{beta}-arrestins 1 and 2 are intracellular adaptor proteins that regulate the subcellular localisation and signalling of GPCRs including Parathyroid Hormone Receptor 1 (PTH1R), which produces {beta}-arrestin-dependent signalling that may selectively stimulate bone anabolism. {beta}-arrestins can themselves be regulated by post-translational modifications including phosphorylation, which dictates the translocation and signalling of the few GPCRs studied in this context to date. We aimed to investigate whether phosphorylation of {beta}-arrestins modulates their subcellular translocation in response to PTH1R activation. Translocation of {beta}-arrestins to the plasma membrane and early endosomes was quantified using BRET biosensors and immunocytochemistry with high content analysis. Phosphoknockout or phosphomimetic mutations were introduced to previously demonstrated phosphorylation sites in {beta}-arrestins 1 and 2, initially in bulk to assess overall effects. Plasma membrane translocation was altered only subtly, whereas early endosome translocation was markedly reduced for both mutations but to a greater degree for the phosphomimetic. Contributions of individual or paired sites were assessed by restoration to wildtype from the bulk phosphomimetic mutants. Dual restoration of S13 and T275 in {beta}-arrestin 1, or the analogous sites in {beta}-arrestin 2, restored the greatest degrees of early endosomal translocation, with complete restoration for {beta}-arrestin 1 and partial for {beta}-arrestin 2. Restoration of other sites had minimal or no effect. Dephosphorylation therefore appears to be required for PTH1R-stimulated {beta}-arrestin early endosomal translocation, with different phosphorylation sites implicated than some other GPCRs. These findings raise the possibility of differential {beta}-arrestin regulation by distinct stimuli which may have implications in therapeutic targeting of {beta}-arrestin-dependent pathways.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Tackling developmental changes in long-term potentiation (LTP) expression during adolescence is crucial to understand disease susceptibility in neurodevelopmental disorders, epileptogenesis or drug abuse. This paper investigated the alterations in the resting phosphorylation state of synaptic proteins essential for synaptic transmission and hippocampal LTP expression from weaning (3 weeks) to adulthood (12 weeks). Synaptic AMPA GluA1 and GluA2 subunit levels increased, yet to different extents, as the GluA1/GluA2 ratio also increased. Conversely, GluA1 phosphorylation in both Ser831 and Ser845 progressively decreased from weaning to adulthood, suggesting an enhanced availability of these sites for activity-dependent phosphorylation. In accordance, LTP induced by different theta-burst stimulation (TBS) intensities in the CA1 area of rat hippocampal slices enhanced gradually in this developmental period. In contrast, basal phosphorylation of GluN1, GluN2B and CaMKII increased during adolescence. Altogether, these findings suggest that alterations in AMPA receptor subunit composition and basal phosphorylation are crucial for the maturation of hippocampal LTP during adolescence, while a mild enhancement in synaptic CaMKII levels may further provide the necessary structural support to LTP expression and stability. Given the reported involvement of GluA1 phosphorylation changes in epileptogenesis and neurodevelopmental disorders, these findings provide important insights into hippocampal synaptic plasticity in normal and altered brain development and epileptogenesis.

Lysophosphatidic acid (LPA) is widely recognized as an extracellular lipid mediator; however, the functional significance of intracellularly produced LPA remains poorly understood. Here, we investigated the regulatory mechanism and functional role of a LPA-producing lysophospholipase D GDE4, also known as GDPD1, in prostate cancer cells. GDE4 expression is induced under ER stress conditions in a PERK-dependent manner and requires the transcription factor ATF3. Disruption of GDE4 expression resulted in altered intracellular levels of LPA and LPA precursor lysophosphatidylethanolamine, accompanied by reduced cell proliferation. RNA sequencing and subsequent validation identified a set of genes downregulated in GDE4-depleted cells. Pharmacological inhibition experiments indicated that peroxisome proliferator-activated receptor and {gamma} (PPAR and PPAR{gamma}) signaling pathways contribute to the regulation of these GDE4-dependent genes. Collectively, our findings suggest that GDE4-dependent lipid remodeling is associated with PPAR/{gamma}-mediated transcriptional regulation under ER stress conditions. These results provide a potential framework for understanding the link between intracellular lipid metabolism and stress-responsive gene regulation.

Highly reactive aldehydes are generated during metabolic processes in the body, and their detoxification is essential for maintaining cellular homeostasis. Hexadecenal, a long-chain fatty aldehyde, is formed during the sphingolipid degradation pathway from the lipid mediator sphingosine-1-phosphate (S1P). However, the cytotoxicity resulting from dysregulation of hexadecenal metabolism is still unclear. To elucidate the effects of impaired hexadecenal metabolism, we analyzed the function of ALDH3A2, an aldehyde dehydrogenase in humans. Our results revealed that ALDH3A2 enzymatic activity is crucial for the suppression of DNA damage, particularly interstrand DNA crosslinks, upon S1P exposure. Furthermore, we demonstrated that hexadecenal accumulation promotes cell death accompanied by the activation of cellular stress responses and morphological abnormalities in the endoplasmic reticulum. These findings suggest that ALDH3A2 functions as a metabolic safeguard to suppress DNA damage and cell death in response to the enhanced metabolic flux of hexadecenal.

RASAL3 acts as a negative regulator of small cellular GTPases in hematopoietic cells. In immune cells, it primarily modulates the RAS/MAPK signaling pathway and affects cellular events including proliferation, differentiation, survival, and migration. Due to its inhibitory role in T cells, RASAL3 may represent a potential modulatory target for improving therapeutic strategies such as cell-based immunotherapy. However, most existing knowledge about RASAL3 function is derived from murine models, and its role in human T-cell signaling remains insufficiently characterized. To address this gap, we systematically investigated the function of RASAL3 in human primary T cells and T-cell line. For this purpose, we employed RASAL3 overexpression, CRISPR/Cas9-mediated deletion, and siRNA-mediated knockdown to thoroughly analyze the effects of RASAL3 on T-cell signaling, proliferation, and migration. Our data demonstrate that RASAL3 modulates primarily CDC42 and RAC1/RAC2 GTPases activity, SAPK/JNK phosphorylation, c-Fos and c-Jun expression, and IL-2 gene promoter activation. In addition, RASAL3 regulates actin polymerization and T-cell migration. Notably, loss of RASAL3 increases Jurkat T cells motility in vivo and potentiates their homing to the spleen. Collectively, these findings identify RASAL3 as an important regulator of human T-cell activation and motility and highlight its application potential for improving CAR-T cell therapy.

NF-{kappa}B essential modulator (NEMO) is the critical scaffold protein of the canonical I kappa B kinase (IKK) complex. Whilst studying the contribution of IKK and IKK{beta} to NF-{kappa}B signalling we noted that NEMO protein levels were reduced in IKK knockout (KO) HCT116 cells. Loss of NEMO was more pronounced in IKK KO cells than IKK{beta} KO, whilst IKK+IKK{beta} double knockout (DKO) cells exhibited a 90% reduction in NEMO abundance. In contrast, the abundance of IKKs was unaffected by NEMO KO, although this abolished canonical NF-{kappa}B activation. Loss of IKKs had no effect on NEMO mRNA levels but IKK DKO cells exhibited accelerated NEMO protein turnover that was reversed by the proteasome inhibitor, MG132, but not the NEDDylation inhibitor, MLN4924. TAK-243, a selective inhibitor of UBA1, the E1 ubiquitin-activating enzyme, increased basal NEMO levels in DKO cells but did not prevent NEMO turnover suggesting both ubiquitylation-dependent and independent pathways for degradation. Bioinformatic analysis identified substantial regions of intrinsic disorder within the N- and C-termini of NEMO. NEMO was rapidly degraded by isolated 20S proteasomes in vitro and this was inhibited by interaction with IKK or IKK{beta}. Finally, re-expression of IKK increased NEMO expression in IKK DKO cells and this required the C-terminal NEMO binding domain (NBD) but not a functional kinase domain. Thus the IKKs act as critical kinase-independent scaffolds that stabilise NEMO; loss of these interactions results in NEMO degradation via the proteasome. These results may be relevant to strategies for inhibiting NF-{kappa}B signalling by disrupting NEMO:IKK interactions.

The transport of about 70 enzymes to lysosomes depends on mannose 6-phosphate signals formed by GNPTAB. Editing of Gnptab in an intestinal mouse cell line revealed the loss of multiple lysosomal enzymes associated with the accumulation of sphingomyelins, ceramides, and cholesterol, and reduced levels of bis(monoacylglycero)phosphate (BMP) in lysosomal proteomes and lipidomes. By cross-correlation we identified two subsets of lysosomal lipid-modifying enzymes linked with mixed unsaturated or di-monosaturated BMP. Autophagy-related proteins functioning in early stages of autophagosome formation associated with neutral ceramides, whereas proteins involved in autophagosome-lysosome fusion correlated with negatively charged BMPs. Therefore, the high cholesterol and low BMP level of lysosomes might be causal for impaired autophagic flux in GNPTAB deficient cells. We propose a functional axis of three lysosomal proteins as potential target to improve the autophagic flux in cells with dysfunctional lysosomes and in a newly established intestinal organoid model suitable as novel experimental tool.