bims-lypmec Biomed News
on Lysosomal positioning and metabolism in cardiomyocytes
Issue of 2024–02–11
eight papers selected by
Satoru Kobayashi, New York Institute of Technology
  1. The GATOR2 complex maintains lysosomal-autophagic function by inhibiting the protein degradation of MiT/TFEs.
  2. Role of the Cytosolic Domain of the a3 Subunit of V-ATPase in the Interaction with Rab7 and Secretory Lysosome Trafficking in Osteoclasts.
  3. Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore.
  4. Rab32 family proteins regulate autophagosomal components recycling.
  5. Lysosomal stress drives the release of pathogenic α-synuclein from macrophage lineage cells via the LRRK2-Rab10 pathway.
  6. The Dance of Proteostasis and Metabolism: Unveiling the Caloristatic Controlling Switch.
  7. Contact-FP: A Dimerization-Dependent Fluorescent Protein Toolkit for Visualizing Membrane Contact Site Dynamics.
  8. AMPK phosphorylation of FNIP1 (S220) controls mitochondrial function and muscle fuel utilization during exercise.
  1. Mol Cell. 2024 Jan 31. pii: S1097-2765(24)00048-0. [Epub ahead of print]
    The GATOR2 complex maintains lysosomal-autophagic function by inhibiting the protein degradation of MiT/TFEs.
    Shu Yang, Chun-Yuan Ting, Mary A Lilly.
      Lysosomes are central to metabolic homeostasis. The microphthalmia bHLH-LZ transcription factors (MiT/TFEs) family members MITF, TFEB, and TFE3 promote the transcription of lysosomal and autophagic genes and are often deregulated in cancer. Here, we show that the GATOR2 complex, an activator of the metabolic regulator TORC1, maintains lysosomal function by protecting MiT/TFEs from proteasomal degradation independent of TORC1, GATOR1, and the RAG GTPase. We determine that in GATOR2 knockout HeLa cells, members of the MiT/TFEs family are ubiquitylated by a trio of E3 ligases and are degraded, resulting in lysosome dysfunction. Additionally, we demonstrate that GATOR2 protects MiT/TFE proteins in pancreatic ductal adenocarcinoma and Xp11 translocation renal cell carcinoma, two cancers that are driven by MiT/TFE hyperactivation. In summary, we find that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.
    Keywords:  E3 ligases; GATOR2; MiT/TFEs; TORC1; autophagy; lysosome; pancreatic ductal adenocarcinoma; renal cell carcinoma; ubiquitination
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.012
  2. Biol Pharm Bull. 2024 ;47(1): 339-344
    Role of the Cytosolic Domain of the a3 Subunit of V-ATPase in the Interaction with Rab7 and Secretory Lysosome Trafficking in Osteoclasts.
    Mayumi Nakanishi-Matsui, Naomi Matsumoto, Ge-Hong Sun-Wada, Yoh Wada.
      We previously reported that the a3 subunit of proton-pumping vacuolar-type ATPase (V-ATPase) interacts with Rab7 and its guanine nucleotide exchange factor, Mon1a-Ccz1, and recruits them to secretory lysosomes in osteoclasts, which is essential for anterograde trafficking of secretory lysosomes. The a3 subunit interacts with Mon1a-Ccz1 through its cytosolic N-terminal domain. Here, we examined the roles of this domain in the interaction with Rab7 and trafficking of secretory lysosomes. Immunoprecipitation experiments showed that a3 interacted with Rab7 through its cytosolic domain, similar to the interaction with Mon1a-Ccz1. We connected this domain with a lysosome localization signal and expressed it in a3-knockout (a3KO) osteoclasts. Although the signal connected to the cytosolic domain was mainly detected in lysosomes, impaired lysosome trafficking in a3KO osteoclasts was not rescued. These results indicate that the cytosolic domain of a3 can interact with trafficking regulators, but is insufficient to induce secretory lysosome trafficking. The C-terminal domain of a3 and other subunits of V-ATPase are likely required to form a fully functional complex for secretory lysosome trafficking.
    Keywords:  Rab7; a3 isoform; osteoclast; secretory lysosome; vacuolar-type ATPase
    DOI:  https://doi.org/10.1248/bpb.b23-00833
  3. Sci Adv. 2024 Feb 09. 10(6): eadj8027
    Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore.
    Shanlin Rao, Marvin Skulsuppaisarn, Lisa M Strong, Xuefeng Ren, Michael Lazarou, James H Hurley, Gerhard Hummer.
      The covalent attachment of ubiquitin-like LC3 proteins (microtubule-associated proteins 1A/1B light chain 3) prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligaseautophagy-related 12 (ATG12)-ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (i) the phosphatidylinositol 3-phosphate effector protein WD repeat domain phosphoinositide-interacting protein 2 (WIPI2), (ii) helix α2 of ATG16L1, and (iii) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12-ATG5-ATG16L1 machinery establishes a high level of regulatory control.
    DOI:  https://doi.org/10.1126/sciadv.adj8027
  4. J Cell Biol. 2024 Mar 04. pii: e202306040. [Epub ahead of print]223(3):
    Rab32 family proteins regulate autophagosomal components recycling.
    Zhe Wu, Huilin Que, Chuangpeng Li, Li Yan, Shixuan Wang, Yueguang Rong.
      In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.
    DOI:  https://doi.org/10.1083/jcb.202306040
  5. iScience. 2024 Feb 16. 27(2): 108893
    Lysosomal stress drives the release of pathogenic α-synuclein from macrophage lineage cells via the LRRK2-Rab10 pathway.
    Tetsuro Abe, Tomoki Kuwahara, Shoichi Suenaga, Maria Sakurai, Sho Takatori, Takeshi Iwatsubo.
      α-Synuclein and LRRK2 are associated with both familial and sporadic Parkinson's disease (PD), although the mechanistic link between these two proteins has remained elusive. Treating cells with lysosomotropic drugs causes the recruitment of LRRK2 and its substrate Rab10 onto overloaded lysosomes and induces extracellular release of lysosomal contents. Here we show that lysosomal overload elicits the release of insoluble α-synuclein from macrophages and microglia loaded with α-synuclein fibrils. This release occurred specifically in macrophage lineage cells, was dependent on the LRRK2-Rab10 pathway and involved exosomes. Also, the uptake of α-synuclein fibrils enhanced the LRRK2 phosphorylation of Rab10, which was accompanied by an increased recruitment of LRRK2 and Rab10 onto lysosomal surface. Our data collectively suggest that α-synuclein fibrils taken up in lysosomes activate the LRRK2-Rab10 pathway, which in turn upregulates the extracellular release of α-synuclein aggregates, leading to a vicious cycle that could enhance α-synuclein propagation in PD pathology.
    Keywords:  Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2024.108893
  6. Cell Stress Chaperones. 2024 Feb 06. pii: S1355-8145(24)00050-6. [Epub ahead of print]
    The Dance of Proteostasis and Metabolism: Unveiling the Caloristatic Controlling Switch.
    Helena Trevisan Schroeder, Carlos Henrique de Lemos Muller, Thiago Gomes Heck, Mauricio Krause, Paulo Ivo Homem de Bittencourt.
      The heat shock response (HSR) is an ancient and evolutionarily conserved mechanism designed to restore cellular homeostasis following proteotoxic challenges. However, it has become increasingly evident that disruptions in energy metabolism also trigger the HSR. This interplay between proteostasis and energy regulation is rooted in the fundamental need for ATP to fuel protein synthesis and repair, making the HSR an essential component of cellular energy management. Recent findings suggest that the origins of proteostasis-defending systems can be traced back over 3.6 billion years, aligning with the emergence of sugar kinases that optimized glycolysis around 3.594 billion years ago. This evolutionary connection is underscored by the spatial similarities between the nucleotide-binding domain of HSP70, the key player in protein chaperone machinery, and hexokinases. The HSR serves as a hub that integrates energy metabolism and resolution of inflammation, further highlighting its role in maintaining cellular homeostasis. Notably, 5'-AMP-activated protein kinase (AMPK) emerges as a central regulator, promoting the HSR during predominantly proteotoxic stress while suppressing it in response to predominantly metabolic stress. The complex relationship between AMPK and the HSR is finely tuned, with paradoxical effects observed under different stress conditions. This delicate equilibrium, known as caloristasis, ensures that cellular homeostasis is maintained despite shifting environmental and intracellular conditions. Understanding the caloristatic controlling switch at the heart of this interplay is crucial. It offers insights into a wide range of conditions, including glycemic control, obesity, type 2 diabetes, cardiovascular and neurodegenerative diseases, reproductive abnormalities, and the optimization of exercise routines. These findings highlight the profound interconnectedness of proteostasis and energy metabolism in cellular function and adaptation.
    Keywords:  5’-AMP-Activated Protein Kinase (AMPK); Caloristasis; Cellular Homeostasis; Energy Metabolism; Heat Shock Response (HSR); Proteostasis
    DOI:  https://doi.org/10.1016/j.cstres.2024.02.002
  7. Contact (Thousand Oaks). 2024 Jan-Dec;7:7 25152564241228911
    Contact-FP: A Dimerization-Dependent Fluorescent Protein Toolkit for Visualizing Membrane Contact Site Dynamics.
    Gregory E Miner, Sidney Y Smith, Wendy K Showalter, Christina M So, Joey V Ragusa, Alex E Powers, Maria Clara Zanellati, Chih-Hsuan Hsu, Michelle F Marchan, Sarah Cohen.
      Membrane contact sites (MCSs) are sites of close apposition between two organelles used to exchange ions, lipids, and information. Cells respond to changing environmental or developmental conditions by modulating the number, extent, or duration of MCSs. Because of their small size and dynamic nature, tools to study the dynamics of MCSs in live cells have been limited. Dimerization-dependent fluorescent proteins (ddFPs) targeted to organelle membranes are an ideal tool for studying MCS dynamics because they reversibly interact to fluoresce specifically at the interface between two organelles. Here, we build on previous work using ddFPs as sensors to visualize the morphology and dynamics of MCSs. We engineered a suite of ddFPs called Contact-FP that targets ddFP monomers to lipid droplets (LDs), the endoplasmic reticulum (ER), mitochondria, peroxisomes, lysosomes, plasma membrane, caveolae, and the cytoplasm. We show that these probes correctly localize to their target organelles. Using LDs as a test case, we demonstrate that Contact-FP pairs specifically localize to the interface between two target organelles. Titration of LD-mitochondria ddFPs revealed that these sensors can be used at high concentrations to drive MCSs or can be titrated down to minimally perturb and visualize endogenous MCSs. We show that Contact-FP probes can be used to: (1) visualize LD-mitochondria MCS dynamics, (2) observe changes in LD-mitochondria MCS dynamics upon overexpression of PLIN5, a known LD-mitochondrial tether, and (3) visualize two MCSs that share one organelle simultaneously (e.g., LD-mitochondria and LD-ER MCSs). Contact-FP probes can be optimized to visualize MCSs between any pair of organelles represented in the toolkit.
    Keywords:  biosensors; caveolae; endoplasmic reticulum; fluorescent proteins; lipid droplets; lysosomes; membrane contact sites; mitochondria; organelles; peroxisomes; plasma membrane
    DOI:  https://doi.org/10.1177/25152564241228911
  8. Sci Adv. 2024 Feb 09. 10(6): eadj2752
    AMPK phosphorylation of FNIP1 (S220) controls mitochondrial function and muscle fuel utilization during exercise.
    Liwei Xiao, Yujing Yin, Zongchao Sun, Jing Liu, Yuhuan Jia, Likun Yang, Yan Mao, Shujun Peng, Zhifu Xie, Lei Fang, Jingya Li, Xiaoduo Xie, Zhenji Gan.
      Exercise-induced activation of adenosine monophosphate-activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle-specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1-transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.
    DOI:  https://doi.org/10.1126/sciadv.adj2752
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