bims-lypmec 2022-10-23 papers

Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2205492119

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes.

Jillian H Kluss, Alexandra Beilina, Chad D Williamson, Patrick A Lewis, Mark R Cookson, Luis Bonet-Ponce.

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Keywords: JIP4; LLOMe; LYTL; Parkinson's disease; kinase

DOI: https://doi.org/10.1073/pnas.2205492119

Nat Commun. 2022 Oct 20. 13(1): 6212

Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo.

Jasjot Singh, Hadeer Elhabashy, Pathma Muthukottiappan, Markus Stepath, Martin Eisenacher, Oliver Kohlbacher, Volkmar Gieselmann, Dominic Winter.

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyze lysosomes and early endosomes. Based on the identification of 5376 cross-links, we investigate protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and a heterodimeric structure of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identify >300 putative cargo proteins and confirm eleven substrates for flotillin-dependent endocytosis, including the latrophilin family of adhesion G protein-coupled receptors.

DOI: https://doi.org/10.1038/s41467-022-33951-0

Front Cell Neurosci. 2022 ;16 895750

Zinc enhances autophagic flux and lysosomal function through transcription factor EB activation and V-ATPase assembly.

Ki-Ryeong Kim, Sang Eun Park, Ji-Ye Hong, Jae-Young Koh, Dong-Hyung Cho, Jung Jin Hwang, Yang-Hee Kim.

The stimulation of autophagy or lysosomes has been considered therapeutic for neurodegenerative disorders because the accumulation of misfolded proteins is commonly observed in the brains of individuals with these diseases. Although zinc is known to play critical roles in the functions of lysosomes and autophagy, the mechanism behind this regulatory relationship remains unclear. Therefore, in this study, we examined which mechanism is involved in zinc-mediated activation of autophagy and lysosome. Exposure to zinc at a sub-lethal concentration activated autophagy in a concentration-dependent manner in mRFP-GFP-LC3-expressing H4 glioma cells. Zinc also rescued the blocking of autophagic flux arrested by pharmaceutical de-acidification. Co-treatment with zinc attenuated the chloroquine (CQ)-induced increase in the number and size of mRFP-GFP-LC3 puncta in H4 cells and accumulation of p62 by CQ or ammonium chloride in both H4 and mouse cerebrocortical cultures. Zinc rapidly induced the expression of cathepsin B (CTSB) and cathepsin D (CTSD), representative lysosomal proteases in neurons, which appeared likely to be mediated by transcription factor EB (TFEB). We observed the translocation of TFEB from neurite to nucleus and the dephosphorylation of TFEB by zinc. The addition of cycloheximide, a chemical inhibitor of protein synthesis, inhibited the activity of CTSB and CTSD at 8 h after zinc exposure but not at 1 h, indicating that only late lysosomal activation was dependent on the synthesis of CTSB and CTSD proteins. At the very early time point, the activation of cathepsins was mediated by an increased assembly of V-ATPase on lysosomes and resultant lysosomal acidification. Finally, considering that P301L mutation in tau protein causes frontotemporal dementia through aggressive tau accumulation, we investigated whether zinc reduces the accumulation of protein aggregates in SK-N-BE(2)-C neuroblastoma cells expressing wild-type tau or mutant P301L-tau. Zinc markedly attenuated the levels of phosphorylated tau and total tau as well as p62 in both wild-type and mutant tau-overexpressing cells. We also observed that zinc was more effective than rapamycin at inducing TFEB-dependent CTSB and CTSD expression and V-ATPase-dependent lysosomal acidification and CTSB/CTSD activation. These results suggest that the regulation of zinc homeostasis could be a new approach for developing treatments for neurodegenerative diseases, including Alzheimer's and Parkinson's.

Keywords: TFEB; V-ATPase; autophagy; cathepsin B; cathepsin D; lysosome; neurodegenerative disease; zinc

DOI: https://doi.org/10.3389/fncel.2022.895750

Oxid Med Cell Longev. 2022 ;2022 7346699

Role of AMPK in Myocardial Ischemia-Reperfusion Injury-Induced Cell Death in the Presence and Absence of Diabetes.

Nirupama Kandula, Saurabh Kumar, Venkata Kiran Kumar Mandlem, Aneela Siddabathuni, Sanjay Singh, Ramoji Kosuru.

Recent studies indicate cell death is the hallmark of cardiac pathology in myocardial infarction and diabetes. The AMP-activated protein kinase (AMPK) signalling pathway is considered a putative salvaging phenomenon, plays a decisive role in almost all cellular, metabolic, and survival functions, and therefore entails precise regulation of its activity. AMPK regulates various programmed cell death depending on the stimuli and context, including autophagy, apoptosis, necroptosis, and ferroptosis. There is substantial evidence suggesting that AMPK is down-regulated in cardiac tissues of animals and humans with type 2 diabetes or metabolic syndrome compared to non-diabetic control and that stimulation of AMPK (physiological or pharmacological) can ameliorate diabetes-associated cardiovascular complications, such as myocardial ischemia-reperfusion injury. Furthermore, AMPK is an exciting therapeutic target for developing novel drug candidates to treat cell death in diabetes-associated myocardial ischemia-reperfusion injury. Therefore, in this review, we summarized how AMPK regulates autophagic, apoptotic, necroptotic, and ferroptosis pathways in the context of myocardial ischemia-reperfusion injury in the presence and absence of diabetes.

DOI: https://doi.org/10.1155/2022/7346699

J Cell Biol. 2022 11 07. pii: e202210014. [Epub ahead of print]221(11):

A second chance at yeast early endosomes.

Allyson F O'Donnell.

Post-endocytic recycling in yeast has been posited to transit solely through the Golgi, raising the possibility that yeast lack early endosomes. In this issue, Laidlaw and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109137) describe a yeast endosomal recycling pathway that gives proteins a second chance to return to the plasma membrane.

DOI: https://doi.org/10.1083/jcb.202210014