bims-lypmec 2025-01-05 papers

Autophagy. 2025 Jan 02. 1-21

Linear ubiquitination at damaged lysosomes induces local NFKB activation and controls cell survival.

Laura Zein, Marvin Dietrich, Denise Balta, Verian Bader, Christoph Scheuer, Suzanne Zellner, Nadine Weinelt, Julia Vandrey, Muriel C Mari, Christian Behrends, Friederike Zunke, Konstanze F Winklhofer, Sjoerd J L Van Wijk.

Lysosomes are the major cellular organelles responsible for nutrient recycling and degradation of cellular material. Maintenance of lysosomal integrity is essential for cellular homeostasis and lysosomal membrane permeabilization (LMP) sensitizes toward cell death. Damaged lysosomes are repaired or degraded via lysophagy, during which glycans, exposed on ruptured lysosomal membranes, are recognized by galectins leading to K48- and K63-linked poly-ubiquitination (poly-Ub) of lysosomal proteins followed by recruitment of the macroautophagic/autophagic machinery and degradation. Linear (M1) poly-Ub, catalyzed by the linear ubiquitin chain assembly complex (LUBAC) E3 ligase and removed by OTULIN (OTU deubiquitinase with linear linkage specificity) exerts important functions in immune signaling and cell survival, but the role of M1 poly-Ub in lysosomal homeostasis remains unexplored. Here, we demonstrate that L-leucyl-leucine methyl ester (LLOMe)-damaged lysosomes accumulate M1 poly-Ub in an OTULIN- and K63 Ub-dependent manner. LMP-induced M1 poly-Ub at damaged lysosomes contributes to lysosome degradation, recruits the NFKB (nuclear factor kappa B) modulator IKBKG/NEMO and locally activates the inhibitor of NFKB kinase (IKK) complex to trigger NFKB activation. Inhibition of lysosomal degradation enhances LMP- and OTULIN-regulated cell death, indicating pro-survival functions of M1 poly-Ub during LMP and potentially lysophagy. Finally, we demonstrate that M1 poly-Ub also occurs at damaged lysosomes in primary mouse neurons and induced pluripotent stem cell-derived primary human dopaminergic neurons. Our results reveal novel functions of M1 poly-Ub during lysosomal homeostasis, LMP and degradation of damaged lysosomes, with important implications for NFKB signaling, inflammation and cell death.Abbreviation: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CRISPR: clustered regularly interspaced short palindromic repeats; CHUK/IKKA: component of inhibitor of nuclear factor kappa B kinase complex; CUL4A-DDB1-WDFY1: cullin 4A-damage specific DNA binding protein 1-WD repeat and FYVE domain containing 1; DGCs: degradative compartments; DIV: days in vitro; DUB: deubiquitinase/deubiquitinating enzyme; ELDR: endo-lysosomal damage response; ESCRT: endosomal sorting complex required for transport; FBXO27: F-box protein 27; GBM: glioblastoma multiforme; IKBKB/IKKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; IKK: inhibitor of NFKB kinase; iPSC: induced pluripotent stem cell; KBTBD7: kelch repeat and BTB domain containing 7; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LCD: lysosomal cell death; LGALS: galectin; LMP: lysosomal membrane permeabilization; LLOMe: L-leucyl-leucine methyl ester; LOP: loperamide; LUBAC: linear ubiquitin chain assembly complex; LRSAM1: leucine rich repeat and sterile alpha motif containing 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; NFKBIA/IĸBα: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; OPTN: optineurin; ORAS: OTULIN-related autoinflammatory syndrome; OTULIN: OTU deubiquitinase with linear linkage specificity; RING: really interesting new gene; RBR: RING-in-between-RING; PLAA: phospholipase A2 activating protein; RBCK1/HOIL-1: RANBP2-type and C3HC4-type zinc finger containing 1; RNF31/HOIP: ring finger protein 31; SHARPIN: SHANK associated RH domain interactor; SQSTM1/p62: sequestosome 1; SR-SIM: super-resolution-structured illumination microscopy; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TH: tyrosine hydroxylase; TNF/TNFα: tumor necrosis factor; TNFRSF1A/TNFR1-SC: TNF receptor superfamily member 1A signaling complex; TRIM16: tripartite motif containing 16; Ub: ubiquitin; UBE2QL1: ubiquitin conjugating enzyme E2 QL1; UBXN6/UBXD1: UBX domain protein 6; VCP/p97: valosin containing protein; WIPI2: WD repeat domain, phosphoinositide interacting 2; YOD1: YOD1 deubiquitinase.

Keywords: Cell death; LUBAC; NF-κB; OTULIN; linear ubiquitination; lysosomes

DOI: https://doi.org/10.1080/15548627.2024.2443945

J Cell Biol. 2025 Mar 03. pii: e202407068. [Epub ahead of print]224(3):

MYO18B promotes lysosomal exocytosis by facilitating focal adhesion maturation.

Wei-Wei Ren, Rebeca Kawahara, Kenichi G N Suzuki, Priya Dipta, Ganglong Yang, Morten Thaysen-Andersen, Morihisa Fujita.

Many cancer cells exhibit increased amounts of paucimannose glycans, which are truncated N-glycan structures rarely found in mammals. Paucimannosidic proteins are proposedly generated within lysosomes and exposed on the cell surface through a yet uncertain mechanism. In this study, we revealed that paucimannosidic proteins are produced by lysosomal glycosidases and secreted via lysosomal exocytosis. Interestingly, lysosomal exocytosis preferentially occurred in the vicinity of focal adhesions, protein complexes connecting the actin cytoskeleton to the extracellular matrix. Through genome-wide knockout screening, we identified that MYO18B, an actin crosslinker, is required for focal adhesion maturation, facilitating lysosomal exocytosis and the release of paucimannosidic lysosomal proteins to the extracellular milieu. Moreover, a mechanosensitive cation channel PIEZO1 locally activated at focal adhesions imports Ca2+ necessary for lysosome-plasma membrane fusion. Collectively, our study unveiled an intimate relationship between lysosomal exocytosis and focal adhesion, shedding light on the unexpected interplay between lysosomal activities and cellular mechanosensing.

DOI: https://doi.org/10.1083/jcb.202407068

Sci Rep. 2025 Jan 02. 15(1): 325

Deficiency in NPC2 results in disruption of mitochondria-late endosome/lysosomes contact sites and endo-lysosomal lipid dyshomeostasis.

Raffaele Pastore, Lihang Yao, Nathan Hatcher, Martin Helley, Janet Brownlees, Radha Desai.

Dysfunction of the endo-lysosomal intracellular Cholesterol transporter 2 protein (NPC2) leads to the onset of Niemann-Pick Disease Type C (NPC), a lysosomal storage disorder. Metabolic and homeostatic mechanisms are disrupted in lysosomal storage disorders (LSDs) hence we characterized a cellular model of NPC2 knock out, to assess alterations in organellar function and inter-organellar crosstalk between mitochondria and lysosomes. We performed characterization of lipid alterations and confirmed altered lysosomal morphology, but no overt changes in oxidative stress markers. Using several techniques, we demonstrated that contacts between mitochondria and late endosomes/lysosomes are reduced in NPC2-/- HEK cells, we observed that the acidic compartments are swollen and lipid dense. Quantification of endogenous lipids in HEKNPC2-/- cells by mass spectrometry reveals accumulation of lipid species indicative of sphingolipid metabolic dysregulation within the lysosome. Specifically, HEK NPC2-/- cells exhibit marked elevation of glucosylsphingosine and glucosylceramides, substrates of beta glucocerebroside (GBA), as well as accumulation of sphingosine and sphingomyelins. Our studies suggest an involvement of NPC2 in the formation of contact sites between mitochondria and lysosomes and support the hypothesis of a role for NPC2 in the endo-lysosomal trafficking pathway and dynamic organellar crosstalk.

DOI: https://doi.org/10.1038/s41598-024-83460-x

Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2409596121

Molecular basis of Spns1-mediated lysophospholipid transport from the lysosome.

Hongwen Chen, Hoa T T Ha, Nadia Elghobashi-Meinhardt, Nhung A Le, Philip Schmiege, Long N Nguyen, Xiaochun Li.

Spns1 mediates the rate-limiting efflux of lysophospholipids from the lysosome to the cytosol. Deficiency of Spns1 is associated with embryonic senescence, as well as liver and skeletal muscle atrophy in animal models. However, the mechanisms by which Spns1 transports lysophospholipid and proton sensing remain unclear. Here, we present a cryogenic electron microscopy structure of human Spns1 in lysophosphatidylcholine (LPC)-bound lumen-facing conformation. Notably, LPC snugly binds within the luminal-open cavity, where the molecular dynamics simulations reveal that LPC presents a propensity to enter between transmembrane-helices (TM) 5 and 8. Structural comparisons and cell-based transport assays uncover several pivotal residues at TM 5/8 that orchestrate the transport cycle, which are unique to Spns1. Furthermore, we identify a five-residue network that is crucial for proton-sensing by Spns1. Transference of these network residues to Spns2, a sphingosine-1-phosphate uniporter, causes the chimeric Spns2 to be low pH dependent. Our results reveal molecular insights into lysosomal LPC transport and the proton-sensing mechanism by Spns1.

Keywords: cryo-EM; lysophospholipids; proton-sensing; transporter

DOI: https://doi.org/10.1073/pnas.2409596121

Int Rev Immunol. 2025 Jan 03. 1-15

E3 ubiquitin ligase Smurf1 promotes cardiomyocyte pyroptosis by mediating ubiquitin-dependent degradation of TRIB2 in a rat model of heart failure.

Wei Liu, Xin Cai, Shiying Duan, Jihua Shen, Jiayuan Wu, Zhengwei Zhou, Kaili Yu, Caihong He, Yuqin Wang.

OBJECTIVE: Heart failure (HF) causes structural and functional changes in the heart, with the pyroptosis-mediated inflammatory response as the core link in HF pathogenesis. E3 ubiquitin ligases participate in cardiovascular disease progression. Here, we explored the underlying molecular mechanisms of E3 ubiquitin ligase Smurf1 in governing HF.
METHODS: HF rat/H9C2 cell models were established by doxorubicin intraperitoneal injections/hypoxia-reoxygenation (H/R), and treated with Smurf1 siRNA and oe-TRIB2 lentivirus plasmids or the NF-κB pathway inhibitor PDTC/si-smurf1, si-TRIB2, protease inhibitor MG132, or lysosomal inhibitor NH4Cl. The cardiac function/cardiac tissue pathological changes/fibrosis in HF rats were evaluated by echocardiography/H&E and Masson staining. GSDMD-N expression was determined by immunohistochemistry. Cell viability/lactate dehydrogenase (LDH) activity/IL-1β and IL-18 levels were measured by CCK-8/LDH kit/ELISA. The interaction between TRIB2 and Smurf1/TRIB2 ubiquitination levels was assessed by co-immunoprecipitation assay. The expression levels of Smurf1 and TRIB2 messenger RNA (mRNA) were determined by RT-qPCR. Levels of Smurf1/TRIB2/the NF-κB pathway-related factors/pyroptosis-related factors and TRIB2 mRNA were determined by Western blot/RT-qPCR.
RESULTS: Smurf1 was highly expressed in H/R-induced H9C2 cells/HF rats, while its knockdown up-regulated TRIB2 and repressed the NF-κB pathway, reduced cardiomyocyte pyroptosis, and attenuated HF. Mechanistically, Smurf1 promoted TRIB2 degradation through an ubiquitin-dependent manner and activated the NF-κB pathway under H/R conditions. TRIB2 silencing annulled Smurf1 knockdown-regulated NF-κB pathway and cardiomyocyte pyroptosis. TRIB2 overexpression inactivated the NF-κB pathway and reduced cardiomyocyte pyroptosis, thus retarding HF.
CONCLUSION: Smurf1 was highly expressed in HF rats, which promoted TRIB2 ubiquitination degradation and activated the NF-κB pathway, thereby promoting cardiomyocyte pyroptosis in HF rats.

Keywords: NF-κB; Smurf1; TRIB2; heart failure; pyroptosis; ubiquitination

DOI: https://doi.org/10.1080/08830185.2024.2434058

Int J Cardiol Cardiovasc Risk Prev. 2024 Dec;23 200338

Diabetic cardiomyopathy: pathophysiology, imaging assessment and therapeutical strategies.

Vincenzo Rizza, Lara Tondi, Angelo Maria Patti, Damiano Cecchi, Massimo Lombardi, Francesco Perone, Marco Ambrosetti, Manfredi Rizzo, Domenico Cianflone, Francesco Maranta.

Diabetes mellitus (DM) is one of the most prevalent cardiovascular risk factors in the general population, being associated with high morbidity and socioeconomic burden. Diabetic cardiomyopathy (DCM) is a non-negligible complication of DM, whose pathophysiological fundaments are the altered cardiac metabolism, the hyperglycemia-triggered formation of advanced glycation end-products (AGEs) and the inflammatory milieu which are typical in diabetic patients. These metabolic abnormalities lead to cardiomyocytes apoptosis, interstitial fibrosis and mechanical cardiac dysfunction, which can be identified with non-invasive imaging techniques, like echocardiography and cardiac magnetic resonance. This review aims to: 1) describe the major imaging features of DCM; 2) highlight how early identification of DCM-related anatomical and functional remodeling might allow patients' therapy optimization and prognosis improvement.

Keywords: Diabetic cardiomyopathy; Heart failure; cardiac imaging; diabetes mellitus

DOI: https://doi.org/10.1016/j.ijcrp.2024.200338

Sci Rep. 2025 Jan 02. 15(1): 422

Two-hit mouse model of heart failure with preserved ejection fraction combining diet-induced obesity and renin-mediated hypertension.

Justin H Berger, Yuji Shi, Timothy R Matsuura, Kirill Batmanov, Xian Chen, Kelly Tam, Mackenzie Marshall, Richard Kue, Jiten Patel, Renee Taing, Russell Callaway, Joanna Griffin, Attila Kovacs, Dinesh Hirenallur-Shanthappa, Russell Miller, Bei B Zhang, Rachel J Roth Flach, Daniel P Kelly.

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We developed a novel "two-hit" model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57Bl6/NJ mice fed a high-fat diet (HFD) for > 10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d. HFD-Renin (aka "HFpEF") mice demonstrated obesity and insulin resistance, moderate left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by echocardiographic measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to human HFpEF. Treatment with empagliflozin, an effective but incompletely understood HFpEF therapy, improved multiple endpoints. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Additional preclinical HFpEF models allow for orthogonal studies to increase validity in assessment of interventions.

Keywords: Cardiovascular disease; Diastolic dysfunction; Heart failure with preserved ejection fraction; Mouse models

DOI: https://doi.org/10.1038/s41598-024-84515-9