bims-lycede 2024-11-10 papers

Mol Cell. 2024 Oct 25. pii: S1097-2765(24)00831-1. [Epub ahead of print]

mTORC1 activity licenses its own release from the lysosomal surface.

Aishwarya Acharya, Constantinos Demetriades.

Nutrient signaling converges on mTORC1, which, in turn, orchestrates a physiological cellular response. A key determinant of mTORC1 activity is its shuttling between the lysosomal surface and the cytoplasm, with nutrients promoting its recruitment to lysosomes by the Rag GTPases. Active mTORC1 regulates various cellular functions by phosphorylating distinct substrates at different subcellular locations. Importantly, how mTORC1 that is activated on lysosomes is released to meet its non-lysosomal targets and whether mTORC1 activity itself impacts its localization remain unclear. Here, we show that, in human cells, mTORC1 inhibition prevents its release from lysosomes, even under starvation conditions, which is accompanied by elevated and sustained phosphorylation of its lysosomal substrate TFEB. Mechanistically, "inactive" mTORC1 causes persistent Rag activation, underlining its release as another process actively mediated via the Rags. In sum, we describe a mechanism by which mTORC1 controls its own localization, likely to prevent futile cycling on and off lysosomes.

Keywords: GATOR1; Rag GTPases; Rheb; TFE3; TFEB; Torin1; lysosomes; mTORC1; rapamycin

DOI: https://doi.org/10.1016/j.molcel.2024.10.008

Mol Cell. 2024 Oct 25. pii: S1097-2765(24)00832-3. [Epub ahead of print]

mTORC1 restricts TFE3 activity by auto-regulating its presence on lysosomes.

Susan Zwakenberg, Denise Westland, Robert M van Es, Holger Rehmann, Jasper Anink, Jolita Ciapaite, Marjolein Bosma, Ellen Stelloo, Nalan Liv, Paula Sobrevals Alcaraz, Nanda M Verhoeven-Duif, Judith J M Jans, Harmjan R Vos, Eleonora Aronica, Fried J T Zwartkruis.

To stimulate cell growth, the protein kinase complex mTORC1 requires intracellular amino acids for activation. Amino-acid sufficiency is relayed to mTORC1 by Rag GTPases on lysosomes, where growth factor signaling enhances mTORC1 activity via the GTPase Rheb. In the absence of amino acids, GATOR1 inactivates the Rags, resulting in lysosomal detachment and inactivation of mTORC1. We demonstrate that in human cells, the release of mTORC1 from lysosomes depends on its kinase activity. In accordance with a negative feedback mechanism, activated mTOR mutants display low lysosome occupancy, causing hypo-phosphorylation and nuclear localization of the lysosomal substrate TFE3. Surprisingly, mTORC1 activated by Rheb does not increase the cytoplasmic/lysosomal ratio of mTORC1, indicating the existence of mTORC1 pools with distinct substrate specificity. Dysregulation of either pool results in aberrant TFE3 activity and may explain nuclear accumulation of TFE3 in epileptogenic malformations in focal cortical dysplasia type II (FCD II) and tuberous sclerosis (TSC).

Keywords: FCD IIb; NPRL2; Rag GTPases; Rheb; TFE3; TSC; amino acids; lysosomes; mTORC1

DOI: https://doi.org/10.1016/j.molcel.2024.10.009

Angew Chem Int Ed Engl. 2024 Nov 04. e202417509

Targeting Lysosomal Thiols for Immunogenic Cancer Cell Death.

Anton Arkhypov, Insa Klemt, Galyna Bila, Dina Attia, Rostyslav Bilyy, Andriy Mokhir.

The number and stability of lysosomes (LYs) are different in cancer and healthy cells that makes them a possible target for cancer specific therapy. However, no LY-targeting drug is clinically approved yet. We describe in this paper a new therapeutic approach based on alkylation of lysosomal thiols in cancer cells by reversible thiol binder 11. The treatment with 11 increases the level of lysosomal reactive oxygen species leading to their destabilization, disruption and immunogenic cancer cell death. These effects are not observed in healthy cells. In murine sarcoma Nemeth-Kellner (NK)/Ly-RB model, 11 exhibits the spectacular therapeutic effect: it extends the lifespan of the treated mice from 21 to 85 days and cures 40% of mice. The survived mice develop antibodies against tumor NK/Ly-RB cells. Their repeated challenge with the NK/Ly-RB cells results in 100% mice survival compared to 0% survival in the control group of naïve mice. Ex vivo data indicate that neutrophils in spleen of the cured animals are also involved in targeting cancer cells and produce neutrophil extracellular traps. In summary, 11 induces the direct antitumor effect supported by humoral immune responses, as well as priming neutrophil's reaction against tumors.

Keywords: Cancer; Electrophilic Drug; Immunogenic Cell Death; Intracellular Thiol; Lysosome

DOI: https://doi.org/10.1002/anie.202417509

J Vis Exp. 2024 Oct 18.

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles.

Shun-Ichi Ito, Yoshinori Tanaka.

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited. To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.

DOI: https://doi.org/10.3791/67385

Sci Rep. 2024 11 06. 14(1): 26875

Lysosomal enzyme binding to the cation-independent mannose 6-phosphate receptor is regulated allosterically by insulin-like growth factor 2.

Richard N Bohnsack, Sandeep K Misra, Jianfang Liu, Mayumi Ishihara-Aoki, Michaela Pereckas, Kazuhiro Aoki, Gang Ren, Joshua S Sharp, Nancy M Dahms.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor's conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

DOI: https://doi.org/10.1038/s41598-024-75300-9