bims-lysosi 2022-08-21 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022–08–21
forty papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly.
AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.
Endocytosis in the axon initial segment maintains neuronal polarity.
AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy.
Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.
TBK1-mTOR Signaling Attenuates Obesity-Linked Hyperglycemia and Insulin Resistance.
Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.
Preprint Highlight: Sugar transporter Slc37a2 regulates bone metabolism via a dynamic tubular lysosomal network in osteoclasts.
TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle.
Covalent JNK inhibitor, JNK-IN-8, suppresses tumor growth in triple-negative breast cancer by activating TFEB and TFE3 mediated lysosome biogenesis and autophagy.
Advances in the genetics and neuropathology of tuberous sclerosis complex: edging closer to targeted therapy.
Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.
Osteoclast rich osteopetrosis due to defects in TCIRG1 gene.
Up-regulation of Osh6 boosts an anti-aging membrane trafficking pathway toward vacuoles.
Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.
Structural basis for proton coupled cystine transport by cystinosin.
Biphasic regulation of osteoblast development via the ERK MAPK-mTOR pathway.
GPNMB confers risk for Parkinson's disease through interaction with α-synuclein.
Presenilin-1 F105C mutation leads to tau accumulation in human neurons via the Akt/mTORC1 signaling pathway.
Chemogenetic activation of hypoglossal motoneurons in a mouse model of Pompe disease.
Anchor Away: A System for Fast Inhibition of Proteins in Drosophila.
Determinants, Maintenance and Function of Organellar pH.
A conserved long-distance telomeric silencing mechanism suppresses mTOR signaling in aging human fibroblasts.
Lipid balance remodelling by human positive-strand RNA viruses and the contribution of lysosomes.
X-chromosomal inactivation patterns in women with Fabry disease.
Endocytic membrane repair by ESCRT-III controls antigen export to the cytosol during antigen cross-presentation.
Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector.
PH domain-mediated autoinhibition and oncogenic activation of Akt.
Biological activity reduction and mitochondrial and lysosomal dysfunction of mesenchymal stem cells aging in vitro.
Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.
MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.
Neuroinflammation in neuronopathic Gaucher disease: Role of microglia and NK cells, biomarkers, and response to substrate reduction therapy.
Rerouting trafficking circuits through posttranslational SNARE modifications.
Structural basis of 3'-end poly(A) RNA recognition by LARP1.
Transcriptome deregulation of peripheral monocytes and whole blood in GBA-related Parkinson's disease.
Double NPY motifs at the N-terminus of the yeast t-SNARE Sso2 synergistically bind Sec3 to promote membrane fusion.
The adaptor protein chaperone AAGAB stabilizes AP-4 complex subunits.
Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells.
Eps15/Pan1p is a master regulator of the late stages of the endocytic pathway.

Nat Commun. 2022 Aug 17. 13(1): 4848

Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly.

Edoardo Ratto, S Roy Chowdhury, Nora S Siefert, Martin Schneider, Marten Wittmann, Dominic Helm, Wilhelm Palm.

Mammalian cells can acquire exogenous amino acids through endocytosis and lysosomal catabolism of extracellular proteins. In amino acid-replete environments, nutritional utilization of extracellular proteins is suppressed by the amino acid sensor mechanistic target of rapamycin complex 1 (mTORC1) through an unknown process. Here, we show that mTORC1 blocks lysosomal degradation of extracellular proteins by suppressing V-ATPase-mediated acidification of lysosomes. When mTORC1 is active, peripheral V-ATPase V1 domains reside in the cytosol where they are stabilized by association with the chaperonin TRiC. Consequently, most lysosomes display low catabolic activity. When mTORC1 activity declines, V-ATPase V1 domains move to membrane-integral V-ATPase Vo domains at lysosomes to assemble active proton pumps. The resulting drop in luminal pH increases protease activity and degradation of protein contents throughout the lysosomal population. These results uncover a principle by which cells rapidly respond to changes in their nutrient environment by mobilizing the latent catabolic capacity of lysosomes.

DOI: https://doi.org/10.1038/s41467-022-32515-6
Nat Commun. 2022 Aug 17. 13(1): 4836

AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.

George K E Umanah, Leire Abalde-Atristain, Mohammed Repon Khan, Jaba Mitra, Mohamad Aasif Dar, Melissa Chang, Kavya Tangella, Amy McNamara, Samuel Bennett, Rong Chen, Vasudha Aggarwal, Marisol Cortes, Paul F Worley, Taekjip Ha, Ted M Dawson, Valina L Dawson.

The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.

DOI: https://doi.org/10.1038/s41467-022-32365-2
Nature. 2022 Aug 17.

Endocytosis in the axon initial segment maintains neuronal polarity.

Kelsie Eichel, Takeshi Uenaka, Vivek Belapurkar, Rui Lu, Shouqiang Cheng, Joseph S Pak, Caitlin A Taylor, Thomas C Südhof, Robert Malenka, Marius Wernig, Engin Özkan, David Perrais, Kang Shen.

Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly1. The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments2,3. How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism that is likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries.

DOI: https://doi.org/10.1038/s41586-022-05074-5
Mol Biol Cell. 2022 Aug 17. mbcE21090473

AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network.

Piyali Majumder, Daisy Edmison, Catherine Rodger, Sruchi Patel, Evan Reid, Swetha Gowrishankar.

The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimer's disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP. Additionally, our results also demonstrate the utility of the human i3Neuronal model system in investigating neuronal phenotypes observed in AP-4 deficient mice and/or the human AP-4 deficiency syndrome. [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E21-09-0473
EMBO J. 2022 Aug 15. e110398

Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy.

Samuel J Rodgers, Emily I Jones, Senthil Arumugam, Sabryn A Hamila, Jill Danne, Rajendra Gurung, Matthew J Eramo, Randini Nanayakkara, Georg Ramm, Meagan J McGrath, Christina A Mitchell.

  Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co-ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient-rich conditions, is unknown. Here, we identify an endosome-dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα-derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation-induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5-kinase, PIKfyve, to generate PI(3,5)P2 . The SNX-BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve-dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient-rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P2 -dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.

Keywords:  INPP4B; PI3Kα; PIKfyve; autophagy; lysosome

DOI:  https://doi.org/10.15252/embj.2021110398
J Biol Chem. 2022 Aug 13. pii: S0021-9258(22)00822-5. [Epub ahead of print] 102379

Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.

Amelia K Luciano, Ekaterina Korobkina, Scott P Lyons, John A Haley, Shelagh Fluharty, Su Myung Jung, Arminja N Kettenbach, David A Guertin.

  Mechanistic Target of Rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2 interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain- and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.

Keywords:  ADP ribosylation factor (ARF); Akt/PKB; BioID2; RICTOR; cell signaling; growth factor; mTORC2; mammalian target of rapamycin (mTOR); proximity labeling

DOI:  https://doi.org/10.1016/j.jbc.2022.102379
Diabetes. 2022 Aug 19. pii: db220256. [Epub ahead of print]

TBK1-mTOR Signaling Attenuates Obesity-Linked Hyperglycemia and Insulin Resistance.

Cagri Bodur, Dubek Kazyken, Kezhen Huang, Aaron Seth Tooley, Kae Won Cho, Tammy M Barnes, Carey N Lumeng, Martin G Myers, Diane C Fingar.

The innate immune kinase TBK1 (TANK-binding kinase 1) responds to microbial-derived signals to initiate responses against viral and bacterial pathogens. More recent work implicates TBK1 in metabolism and tumorigenesis. The kinase mTOR (mechanistic target of rapamycin) integrates diverse environmental cues to control fundamental cellular processes. Our prior work demonstrated in cells that TBK1 phosphorylates mTOR (on S2159) to increase mTORC1 and mTORC2 catalytic activity and signaling. Here we investigate a role for TBK1-mTOR signaling in control of glucose metabolism in vivo. We find that diet induced obese (DIO) but not lean mice bearing a whole-body "TBK1 resistant" Mtor S2159A knockin allele (MtorA/A) display exacerbated hyperglycemia and systemic insulin resistance with no change in energy balance. Mechanistically, Mtor S2159A knockin in DIO mice reduces mTORC1 and mTORC2 signaling in response to insulin and innate immune agonists, reduces anti-inflammatory gene expression in adipose tissue, and blunts anti-inflammatory macrophage M2 polarization, phenotypes shared by mice with tissue-specific inactivation of TBK1 or mTOR complexes. Tissues from DIO mice display elevated TBK1 activity and mTOR S2159 phosphorylation relative to lean mice. We propose a model whereby obesity-associated signals increase TBK1 activity and mTOR phosphorylation, which boosts mTORC1 and mTORC2 signaling in parallel to the insulin pathway, thereby attenuating insulin resistance to improve glycemic control during diet-induced obesity.

DOI: https://doi.org/10.2337/db22-0256
Biochemistry. 2022 Aug 18.

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.

Michael C Yoon, Vivian Hook, Anthony J O'Donoghue.

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

DOI: https://doi.org/10.1021/acs.biochem.2c00358
Mol Biol Cell. 2022 Sep 01. 33(10): mbcP22071005

Preprint Highlight: Sugar transporter Slc37a2 regulates bone metabolism via a dynamic tubular lysosomal network in osteoclasts.

Adriana Mantegazza.

DOI: https://doi.org/10.1091/mbc.P22-07-1005
J Cell Sci. 2022 Aug 17. pii: jcs.260378. [Epub ahead of print]

TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle.

Paolo Guerra, Luc-Alban P E Vuillemenot, Yulan B van Oppen, Marije Been, Andreas Milias-Argeitis.

  Recent studies have revealed that the growth rate of budding yeast and mammalian cells varies during the cell cycle. By linking a multitude of signals to cell growth, the highly conserved Target of Rapamycin Complex 1 (TORC1) and Protein Kinase A (PKA) pathways are prime candidates for mediating the dynamic coupling between growth and division. However, measurements of TORC1 and PKA activity during the cell cycle are still lacking. Following the localization dynamics of two TORC1 and PKA targets via time-lapse microscopy in hundreds of yeast cells, we found that the activity of these pathways towards ribosome biogenesis fluctuates in synchrony with the cell cycle even under constant external conditions. Mutations of upstream TORC1 and PKA regulators suggested that internal metabolic signals partially mediate these activity changes. Our study reveals a new aspect of TORC1 and PKA signaling, which will be important for understanding growth regulation during the cell cycle.

Keywords:  Cell growth; PKA Signaling; Ribosome biogenesis; Single-cell microscopy; TOR signaling; TORC1

DOI:  https://doi.org/10.1242/jcs.260378
Mol Cancer Ther. 2022 Aug 17. pii: MCT-21-1044. [Epub ahead of print]

Covalent JNK inhibitor, JNK-IN-8, suppresses tumor growth in triple-negative breast cancer by activating TFEB and TFE3 mediated lysosome biogenesis and autophagy.

Milad Soleimani, Alexander Somma, Tamer S Kaoud, Ria Goyal, Jorge Bustamante, Dennis C Wylie, Nisha Holay, Agnieszka P Looney, Uma Giri, Todd Triplett, Kevin N Dalby, Jeanne Kowalski, S Gail Eckhardt, Carla Van Den Berg.

The heterogeneity and aggressiveness of Triple-Negative Breast Cancer (TNBC) contribute to its early recurrence and metastasis. Despite substantial research to identify effective therapeutic targets, TNBC remains elusive in terms of improving patient outcomes. Here, we report that a covalent JNK inhibitor, JNK-IN-8, suppresses TNBC growth both in vitro and in vivo. JNK-IN-8 reduced colony formation, cell viability, and organoid growth in vitro and slowed patient-derived xenograft and syngeneic tumor growth in vivo. Cells treated with JNK-IN-8 exhibited large, cytoplasmic vacuoles with lysosomal markers. To examine the molecular mechanism of this phenotype, we looked at the master regulators of lysosome biogenesis and autophagy TFEB and TFE3. JNK-IN-8 inhibited TFEB phosphorylation and induced nuclear translocation of unphosphorylated TFEB and TFE3. This was accompanied by an upregulation of TFEB/TFE3 target genes associated with lysosome biogenesis and autophagy. Depletion of both TFEB and TFE3 diminished the JNK-IN-8-driven upregulation of lysosome biogenesis/autophagy markers. TFEB and TFE3 are phosphorylated by a number of kinases, including mTOR. JNK-IN-8 reduced phosphorylation of mTOR targets in a concentration-dependent manner. Knockout of JNK1 and/or JNK2 had no impact on TFEB/TFE3 activation or mTOR inhibition by JNK-IN-8, but inhibited colony formation. Similarly, re-expression of either wildtype or drug-nonbinding JNK (C116S) in JNK knockout cells did not reverse JNK-IN-8-induced TFEB dephosphorylation. In summary, JNK-IN-8 induced lysosome biogenesis and autophagy by activating TFEB/TFE3 via mTOR inhibition independently of JNK. Together, these findings demonstrate the efficacy of JNK-IN-8 as a targeted therapy for TNBC and reveal its novel lysosome- and autophagy-mediated mechanism of action.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-1044
Lancet Neurol. 2022 09;pii: S1474-4422(22)00213-7. [Epub ahead of print]21(9): 843-856

Advances in the genetics and neuropathology of tuberous sclerosis complex: edging closer to targeted therapy.

Paolo Curatolo, Nicola Specchio, Eleonora Aronica.

Tuberous sclerosis complex is a rare genetic disease associated with mutations in the TSC1 or TSC2 genes, which cause overactivation of the mTOR complex. In the past 5 years, understanding has increased of the cellular consequences of TSC1 and TSC2 genetic variants and the mTORC1 overactivation in neurons and glial cells and their contribution to network dysfunction. Infants and young children (aged 1-5 years) with tuberous sclerosis complex might now benefit from early assessment of gene variant status and mosaicism. In the past 5 years, substantial advances have also been made in our understanding of mTOR-related neuropathology and the molecular aspects of both epileptogenesis and co-occurring neurodevelopmental disorders. Many potential disease-modifying strategies have been identified, including developments in targeted therapies based on molecular findings in epilepsy. Reliable EEG and MRI biomarkers are now available to identify, at a younger age than previously possible, infants with tuberous sclerosis complex who are at risk of epilepsy, autism, and developmental delay. Vigabatrin has been used successfully as a treatment in infants with tuberous sclerosis complex who showed abnormalities on EEG before seizure onset. The scope for mitigation of tuberous sclerosis complex-associated symptoms has expanded, including the use of mTOR inhibitors such as sirolimus and everolimus. Close cooperation between clinical and basic neuroscientists has provided new opportunities for future advances.

DOI: https://doi.org/10.1016/S1474-4422(22)00213-7
Autophagy. 2022 Aug 18. 1-18

Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.

Xiangyu Zhang, Santosh Kumar Misra, Parikshit Moitra, Xiuli Zhang, Se-Jin Jeong, Jeremiah Stitham, Astrid Rodriguez-Velez, Arick Park, Yu-Sheng Yeh, William E Gillanders, Daping Fan, Abhinav Diwan, Jaehyung Cho, Slava Epelman, Irfan J Lodhi, Dipanjan Pan, Babak Razani.

  Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1.

Keywords:  Acidic nanoparticles; PLGA; atherosclerosis; lysosomal dysfunction; macrophage

DOI:  https://doi.org/10.1080/15548627.2022.2108252
Bone. 2022 Aug 15. pii: S8756-3282(22)00196-X. [Epub ahead of print] 116519

Osteoclast rich osteopetrosis due to defects in TCIRG1 gene.

Valentina Capo, Mario Abinun, Anna Villa.

  The discovery that mutations in TCIRG1 (also known as Atp6i) gene are responsible for the majority of autosomal recessive osteopetrosis (ARO) forms in humans heralded a new era for comprehension of this heterogeneous rare bone disease. TCIRG1 gene encodes the a3 subunit, an essential isoform of the vacuolar ATPase proton pump involved in the acidification of the resorption lacuna and in secretory lysosome trafficking. The gene defects lead to inefficient bone resorption by unfunctional osteoclasts which are seen in abundance on bone marrow biopsy, delineating this form of ARO as 'osteoclast-rich'. Patients usually present in early childhood with features of extramedullary haematopoiesis (hepatosplenomegaly, anaemia, thrombocytopenia) due to bone marrow fibrosis, and cranial nerve encroachment (blindness in particular). Impaired gastric calcium uptake due to high pH causes the co-occurrence of rickets, described as "osteopetrorickets". Osteoclast dysfunction leads to early death if untreated and allogeneic haematopoietic stem cell transplantation is the treatment of choice. Combined studies in patients and mouse models carrying spontaneous (the oc/oc mouse) or targeted disruption of Atp6i (TCIRG1) gene have been instrumental for the insight into disease pathogenesis and for the development of novel cellular therapies exploiting gene correction.

Keywords:  Gene therapy; Haematopoietic stem cell transplantation; Osteoclast function; Osteopetrosis; Vacuolar-type proton-pumping ATPase

DOI:  https://doi.org/10.1016/j.bone.2022.116519
Microb Cell. 2022 Aug 01. 9(8): 145-157

Up-regulation of Osh6 boosts an anti-aging membrane trafficking pathway toward vacuoles.

Ilham Kadhim, Nazneen Begum, William King, Licheng Xu, Fusheng Tang.

  Members of the family of oxysterol-binding proteins mediate non-vesicular lipid transport between membranes and contribute to longevity in different manners. We previously found that a 2-fold up-regulation of Osh6, one of seven yeast oxysterol-binding proteins, remedies vacuolar morphology defects in mid-aged cells, partly down-regulates the target of rapamycin complex 1 (TORC1), and increases the replicative lifespan. At the molecular level, Osh6 transports phosphatidylserine (PS) and phosphatidylinositol-4-phosphate (PI4P) between the endoplasmic reticulum (ER) and the plasma membrane (PM). To decipher how an ER-PM working protein controls vacuolar morphology, we tested genetic interactions between OSH6 and DRS2, whose protein flips PS from the lumen to the cytosolic side of the Golgi, the organelle between ER and vacuoles in many pathways. Up-regulated OSH6 complemented vacuolar morphology of drs2Δ and enriched PI4P on the Golgi, indicating that Osh6 also works on the Golgi. This altered PI4P-enrichment led to a delay in the secretion of the proton ATPase Pma1 to the PM and a rerouting of Pma1 to vacuoles in a manner dependent on the trans-Golgi network (TGN) to late endosome (LE) trafficking pathway. Since the TGN-LE pathway controls endosomal and vacuolar TORC1, it may be the anti-aging pathway boosted by up-regulated Osh6.

Keywords:  Golgi; Osh6; PI4P; longevity; vacuole

DOI:  https://doi.org/10.15698/mic2022.08.783
J Immunol. 2022 Aug 19. pii: ji2200280. [Epub ahead of print]

Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

Jia-Feng Cao, Li-Guo Ding, Qing-Chao Wang, Guang-Kun Han, Da-Cheng Qin, Gao-Feng Cheng, Zhao-Ran Dong, Qing-Jiang Mu, Wei-Guang Kong, Xia Liu, Yong-Yao Yu, Zhen Xu.

Mammalian studies have demonstrated that B cell immune responses are regulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling. Teleost fish represent the oldest living bony vertebrates that contain bona fide B cells. So far, whether the regulatory mechanism of mTORC1 signaling in B cells occurred in teleost fish is still unknown. In this study, we developed a fish model by using rapamycin (RAPA) treatment to inhibit mTORC1 signaling and demonstrated the role of mTORC1 signaling in teleost B cells. In support, we found inhibition of mTORC1 signaling by RAPA decreased the phagocytic capacity, proliferation, and Ig production of B cells. Critically, Flavobacterium columnare induced specific IgM binding in serum, and these titers were significantly inhibited by RAPA treatment, thus decreasing Ab-mediated agglutination of F. columnare and significantly increasing the susceptibility of fish upon F. columnare reinfection. Collectively, our findings elucidated that the mTORC1 pathway is evolutionarily conserved in regulating B cell responses, thus providing a new point for understanding the B cells functions in teleost fish.

DOI: https://doi.org/10.4049/jimmunol.2200280
Nat Commun. 2022 Aug 17. 13(1): 4845

Structural basis for proton coupled cystine transport by cystinosin.

Mark Löbel, Sacha P Salphati, Kamel El Omari, Armin Wagner, Stephen J Tucker, Joanne L Parker, Simon Newstead.

Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.

DOI: https://doi.org/10.1038/s41467-022-32589-2
Elife. 2022 Aug 17. pii: e78069. [Epub ahead of print]11

Biphasic regulation of osteoblast development via the ERK MAPK-mTOR pathway.

Jung-Min Kim, Yeon-Suk Yang, Jaehyoung Hong, Sachin Chaugule, Hyonho Chun, Marjolein C H van der Meulen, Ren Xu, Matthew B Greenblatt, Jae-Hyuck Shim.

  Emerging evidence supports that osteogenic differentiation of skeletal progenitors is a key determinant of overall bone formation and bone mass. Despite extensive studies showing the function of mitogen-activated protein kinases (MAPKs) in osteoblast differentiation, none of these studies show in vivo evidence of a role for MAPKs in osteoblast maturation subsequent to lineage commitment. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Map2k1 (MEK1) and Map2k2 (MEK2), in mature osteoblasts and osteocytes, markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts. also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partially reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.

Keywords:  medicine; mouse

DOI:  https://doi.org/10.7554/eLife.78069
Science. 2022 Aug 19. 377(6608): eabk0637

GPNMB confers risk for Parkinson's disease through interaction with α-synuclein.

Maria E Diaz-Ortiz, Yunji Seo, Marijan Posavi, Marc Carceles Cordon, Elisia Clark, Nimansha Jain, Rakshita Charan, Michael D Gallagher, Travis L Unger, Noor Amari, R Tyler Skrinak, Roseanne Davila-Rivera, Eliza M Brody, Noah Han, Rebecca Zack, Vivianna M Van Deerlin, Thomas F Tropea, Kelvin C Luk, Edward B Lee, Daniel Weintraub, Alice S Chen-Plotkin.

Many risk loci for Parkinson's disease (PD) have been identified by genome-wide association studies (GWASs), but target genes and mechanisms remain largely unknown. We linked the GWAS-derived chromosome 7 locus (sentinel single-nucleotide polymorphism rs199347) to GPNMB through colocalization analyses of expression quantitative trait locus and PD risk signals, confirmed by allele-specific expression studies in the human brain. In cells, glycoprotein nonmetastatic melanoma protein B (GPNMB) coimmunoprecipitated and colocalized with α-synuclein (aSyn). In induced pluripotent stem cell-derived neurons, loss of GPNMB resulted in loss of ability to internalize aSyn fibrils and develop aSyn pathology. In 731 PD and 59 control biosamples, GPNMB was elevated in PD plasma, associating with disease severity. Thus, GPNMB represents a PD risk gene with potential for biomarker development and therapeutic targeting.

DOI: https://doi.org/10.1126/science.abk0637
Cell Biosci. 2022 Aug 14. 12(1): 131

Presenilin-1 F105C mutation leads to tau accumulation in human neurons via the Akt/mTORC1 signaling pathway.

Cheong-Meng Chong, Yuan Tan, Jiaqi Tong, Minjing Ke, Ke Zhang, Lingli Yan, Xiaotong Cen, Jia-Hong Lu, Guobing Chen, Huanxing Su, Dajiang Qin.

   BACKGROUND: The mammalian target of rapamycin (mTOR) plays a critical role in controlling cellular homeostasis, and its dysregulation has been implicated in Alzheimer's disease (AD). Presenilin-1 (PS1) mutations account for the most common causes of familial Alzheimer's disease (FAD); however, whether PS1 mutation causes mTOR dysregulation in human neurons remains a key unresolved issue.
METHODS: We generated heterozygotes and homozygotes of PS1 F105C knock-in mutation in human induced pluripotent stem cells (iPSCs) via CRISPR/Cas9/piggyback-based gene editing and differentiated them into human neurons. Secreted Aβ and tau accumulation were determined by ELISA assay, immunofluorescence staining, and western blotting analysis. mTOR signaling was evaluated by western blotting analysis, immunofluorescence staining, and co-immunoprecipitation. Autophagy/lysosome activities were determined by LC3-based assay, LysoTracker Red staining, and DQ-Red BSA staining.
RESULTS: Through comparison among these isogenic neurons, PS1 F105C mutant neurons exhibited elevated Aβ and tau accumulation. In addition, we found that the response of mTORC1 to starvation decreases in PS1 F105C mutant neurons. The Akt/mTORC1/p70S6K signaling pathway remained active upon EBSS starvation, leading to the co-localization of the vast majority of mTOR with lysosomes. Consistently, PS1 F105C neurons displayed a significant decline in starvation-induced autophagy. Notably, Torin1, a mTOR inhibitor, could efficiently reduce prominent tau pathology that occurred in PS1 F105C neurons.
CONCLUSION: We demonstrate that Chinese PS1 F105C mutation causes dysregulation of mTORC1 signaling, contributing to tau accumulation in human neurons. This study on inherited FAD PS1 mutation provides unprecedented insights into our understanding of the molecular mechanisms of AD. It supports that pharmaceutical blocking of mTOR is a promising therapeutic strategy for the treatment of AD.

Keywords:  CRISPR/Cas9; Presenilin-1; iPSC; mTOR

DOI:  https://doi.org/10.1186/s13578-022-00874-8
J Neurophysiol. 2022 Aug 17.

Chemogenetic activation of hypoglossal motoneurons in a mouse model of Pompe disease.

Michele Lynn Singer, Sabhya Rana, Ethan S Benevides, Brian E Barral, Barry J Byrne, David D Fuller.

  Pompe disease is a lysosomal storage disease resulting from absence or deficiency of acid α-glucosidase (GAA). Tongue related disorders including dysarthria, dysphagia, and obstructive sleep apnea are common in Pompe disease. Our purpose was to determine if designer receptors exclusively activated by designer drugs (DREADDs) could be used to stimulate tongue motor output in a mouse model of Pompe disease. An adeno-associated virus serotype 9 (AAV9) encoding an excitatory DREADD (AAV9-hSyn-hM3D(Gq)-mCherry, 2.44 x 1010 vg) was administered to the posterior tongue of 5-7 week old Gaa null (Gaa-/-) mice. Lingual EMG responses to intraperitoneal injection of saline or a DREADD ligand (JHU37160-dihydrochloride, J60) were assessed 12 weeks later during spontaneous breathing. Saline injection produced no consistent changes in lingual EMG. Following the DREADD ligand, there were statistically significant (P<0.05) increases in both tonic and phasic inspiratory EMG activity recorded from the posterior tongue. Brainstem histology confirmed mCherry expression in hypoglossal (XII) motoneurons in all mice, thus verifying retrograde movement of the AAV9 vector. Morphologically, Gaa-/- XII motoneurons showed histologic characteristics that are typical of Pompe disease, including an enlarged soma and vacuolization. We conclude that lingual delivery of AAV9 can be used to drive functional expression of DREADD in XII motoneurons in a mouse model of Pompe disease.

Keywords:  DREADD; Pompe; chemogenetics; hypoglossal; tongue

DOI:  https://doi.org/10.1152/jn.00026.2022
Methods Mol Biol. 2022 ;2540 239-249

Anchor Away: A System for Fast Inhibition of Proteins in Drosophila.

Pablo Sanchez Bosch.

  Anchor away is a sequestering method designed to acutely and timely abrogate the function of a protein of interest by anchoring to a cell compartment different from its target. This method induces the binding of the target protein to the anchor by either the addition of rapamycin to Drosophila food or cell media. Rapamycin mediates the formation of a ternary complex between the anchor, which is tagged with the FK506-binding protein (FKBP12), and the target protein fused with the FKB12 rapamycin-binding (FRB) domain of mammalian target of rapamycin (mTOR). The rapamycin-bound target protein stays sequestered away from its compartment, where it cannot perform its biological function.

Keywords:  CRISPR; Loss of function; Protein knockout; Rapamycin; TOR

DOI:  https://doi.org/10.1007/978-1-0716-2541-5_11
Physiol Rev. 2022 Aug 18.

Determinants, Maintenance and Function of Organellar pH.

Spencer A Freeman, Sergio Grinstein, John Orlowski.

  The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation and functional activity. In addition, protons (H+, or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. While the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the [H+] of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen, together with advances in genomic, proteomic and electrophysiological techniques have led to the identification and characterization of unique pumps, channels and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications to cellular function in health and disease are the subject of this review.

Keywords:  H+-ATPasea; Organelles; cotransporters; exchangers; pH regulation

DOI:  https://doi.org/10.1152/physrev.00009.2022
Sci Adv. 2022 Aug 19. 8(33): eabk2814

A conserved long-distance telomeric silencing mechanism suppresses mTOR signaling in aging human fibroblasts.

Kathrin Jäger, Juliane Mensch, Maria Elisabeth Grimmig, Bruno Neuner, Kerstin Gorzelniak, Seval Türkmen, Ilja Demuth, Alexander Hartmann, Christiane Hartmann, Felix Wittig, Anje Sporbert, Andreas Hermann, Georg Fuellen, Steffen Möller, Michael Walter.

Telomeres are repetitive nucleotide sequences at the ends of each chromosome. It has been hypothesized that telomere attrition evolved as a tumor suppressor mechanism in large long-lived species. Long telomeres can silence genes millions of bases away through a looping mechanism called telomere position effect over long distances (TPE-OLD). The function of this silencing mechanism is unknown. We determined a set of 2322 genes with high positional conservation across replicatively aging species that includes known and candidate TPE-OLD genes that may mitigate potentially harmful effects of replicative aging. Notably, we identified PPP2R2C as a tumor suppressor gene, whose up-regulation by TPE-OLD in aged human fibroblasts leads to dephosphorylation of p70S6 kinase and mammalian target of rapamycin suppression. A mechanistic link between telomeres and a tumor suppressor mechanism supports the hypothesis that replicative aging fulfills a tumor suppressor function and motivates previously unknown antitumor and antiaging strategies.

DOI: https://doi.org/10.1126/sciadv.abk2814
Antiviral Res. 2022 Aug 17. pii: S0166-3542(22)00167-X. [Epub ahead of print] 105398

Lipid balance remodelling by human positive-strand RNA viruses and the contribution of lysosomes.

Michele Lai, Alessandro De Carli, Carolina Filipponi, Elena Iacono, Veronica La Rocca, Giulia Lottini, Carmen Rita Piazza, Paola Quaranta, Maria Sidoti, Mauro Pistello, Giulia Freer.

  A marked reorganization of internal membranes occurs in the cytoplasm of cells infected by single stranded positive-sense RNA viruses. Most cell compartments change their asset to provide lipids for membrane rearrangement into replication organelles, where to concentrate viral proteins and enzymes while hiding from pathogen pattern recognition molecules. Because the endoplasmic reticulum is a central hub for lipid metabolism, when viruses hijack the organelle to form their replication organelles, a cascade of events change the intracellular environment. This results in a marked increase in lipid consumption, both by lipolysis and lipophagy of lipid droplets. In addition, lipids are used to produce energy for viral replication. At the same time, inflammation is started by signalling lipids, where lysosomal processing plays a relevant role. This review is aimed at providing an overview on what takes place after human class IV viruses have released their genome into the host cell and the consequences on lipid metabolism, including lysosomes.

Keywords:  Coronavirus; Flavivirus; Lipid droplet; Lipid metabolism; Lysosome; Replication organelle

DOI:  https://doi.org/10.1016/j.antiviral.2022.105398
Mol Genet Genomic Med. 2022 Aug 16. e2029

X-chromosomal inactivation patterns in women with Fabry disease.

Laura Wagenhäuser, Vanessa Rickert, Claudia Sommer, Christoph Wanner, Peter Nordbeck, Simone Rost, Nurcan Üçeyler.

   BACKGROUND: Although Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA), women may develop severe symptoms. We investigated X-chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women.
PATIENTS AND METHODS: We included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X-chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity.
RESULTS: 43/95 (45%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25% distribution) in 6/87 (7%) mouth epithelial cell samples, 31/88 (35%) blood samples, and 9/27 (33%) skin fibroblast samples. Clinical phenotype, α-galactosidase A (GAL) activity, and lyso-Gb3 levels did not show intergroup differences when stratified for X-chromosomal skewing and activity status of the mutated X-chromosome.
CONCLUSIONS: X-inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns.

Keywords:  Fabry disease; Fabry genotype; Fabry phenotype; X-chromosomal inactivation; female Fabry patients

DOI:  https://doi.org/10.1002/mgg3.2029
Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01022-1. [Epub ahead of print]40(7): 111205

Endocytic membrane repair by ESCRT-III controls antigen export to the cytosol during antigen cross-presentation.

Marine Gros, Elodie Segura, Derek C Rookhuizen, Blandine Baudon, Sandrine Heurtebise-Chrétien, Nina Burgdorf, Mathieu Maurin, Eugene A Kapp, Richard J Simpson, Patrycja Kozik, Jose A Villadangos, Mathieu J M Bertrand, Marianne Burbage, Sebastian Amigorena.

  Despite its crucial role in initiation of cytotoxic immune responses, the molecular pathways underlying antigen cross-presentation remain incompletely understood. The mechanism of antigen exit from endocytic compartments into the cytosol is a long-standing matter of controversy, confronting two main models: transfer through specific channels/transporters or rupture of endocytic membranes and leakage of luminal content. By monitoring the occurrence of intracellular damage in conventional dendritic cells (cDCs), we show that cross-presenting cDC1s display more frequent endomembrane injuries and increased recruitment of endosomal sorting complex required for transport (ESCRT)-III, the main repair system for intracellular membranes, relative to cDC2s. Silencing of CHMP2a or CHMP4b, two effector subunits of ESCRT-III, enhances cytosolic antigen export and cross-presentation. This phenotype is partially reversed by chemical inhibition of RIPK3, suggesting that endocytic damage is related to basal activation of the necroptosis pathway. Membrane repair therefore proves crucial in containing antigen export to the cytosol and cross-presentation in cDCs.

Keywords:  CP: Cell biology; CP: Immunology; ESCRT-III; cross-presentation; dendritic cells; endomembrane disruption; membrane repair; necroptosis

DOI:  https://doi.org/10.1016/j.celrep.2022.111205
Cell Death Discov. 2022 Aug 13. 8(1): 357

Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector.

Ruizhi Li, Kristin F Wilson, Richard A Cerione.

Proliferating cancer cells are dependent on glutamine metabolism for survival when challenged with oxidative stresses caused by reactive oxygen species, hypoxia, nutrient deprivation and matrix detachment. ATF4, a key stress responsive transcription factor, is essential for cancer cells to sustain glutamine metabolism when challenged with these various types of stress. While it is well documented how the ATF4 transcript is translated into protein as a stress response, an important question concerns how the ATF4 message levels are sustained to enable cancer cells to survive the challenges of nutrient deprivation and damaging reactive oxygen species. Here, we now identify the pathway in triple negative breast cancer cells that provides a sustained ATF4 response and enables their survival when encountering these challenges. This signaling pathway starts with mTORC2, which upon sensing cellular stresses arising from glutamine deprivation or an acute inhibition of glutamine metabolism, initiates a cascade of events that triggers an increase in ATF4 transcription. Surprisingly, this signaling pathway is not dependent on AKT activation, but rather requires the mTORC2 target, PKC, which activates the transcription factor Nrf2 that then induces ATF4 expression. Additionally, we identify a sirtuin family member, the NAD+-dependent de-succinylase Sirt5, as a key transcriptional target for ATF4 that promotes cancer cell survival during metabolic stress. Sirt5 plays fundamental roles in supporting cancer cell metabolism by regulating various enzymatic activities and by protecting an enzyme essential for glutaminolysis, glutaminase C (GAC), from degradation. We demonstrate that ectopic expression of Sirt5 compensates for knockdowns of ATF4 in cells exposed to glutamine deprivation-induced stress. These findings provide important new insights into the signaling cues that lead to sustained ATF4 expression as a general stress-induced regulator of glutamine metabolism, as well as highlight Sirt5 an essential effector of the ATF4 response to metabolic stress.

DOI: https://doi.org/10.1038/s41420-022-01156-5
Elife. 2022 Aug 15. pii: e80148. [Epub ahead of print]11

PH domain-mediated autoinhibition and oncogenic activation of Akt.

Hwan Bae, Thibault Viennet, Eunyoung Park, Nam Chu, Antonieta Salguero, Michael J Eck, Haribabu Arthanari, Philip A Cole.

  Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.80148
Stem Cell Res Ther. 2022 Aug 13. 13(1): 411

Biological activity reduction and mitochondrial and lysosomal dysfunction of mesenchymal stem cells aging in vitro.

Ge Zhang, Yuli Wang, Jianhua Lin, Bo Wang, Ali Mohsin, Zhimin Cheng, Weijie Hao, Wei-Qiang Gao, Huiming Xu, Meijin Guo.

   BACKGROUND: Mesenchymal stem cells (MSCs) have been extensively used for the treatment of various diseases in preclinical and clinical trials. In vitro propagation is needed to attain enough cells for clinical use. However, cell aging and viability reduction caused by long-time culture have not been thoroughly investigated, especially for the function of mitochondria and lysosomes. Therefore, this study was designed to detect mitochondrial and lysosomal activity, morphological and functional changes in human umbilical cord MSCs (UMSCs) after long-time culture.
METHODS: First, we examined cell activities, including proliferation and immigration ability, differentiation potential, and immunosuppressive capacity of UMSCs at an early and late passages as P4 (named UMSC-P4) and P9 (named UMSC-P9), respectively. Then, we compared the mitochondrial morphology of UMSC-P4 and UMSC-P9 using the electronic microscope and MitoTracker Red dyes. Furthermore, we investigated mitochondrial function, including mitochondrial membrane potential, antioxidative ability, apoptosis, and ferroptosis detected by respective probe. Cell energy metabolism was tested by mass spectrometry. In addition, we compared the lysosomal morphology of UMSC-P4 and UMSC-P9 by electronic microscope and lysoTracker Red dyes. Finally, the transcriptome sequence was performed to analyze the total gene expression of these cells.
RESULTS: It was found that UMSC-P9 exhibited a reduced biological activity and showed an impaired mitochondrial morphology with disordered structure, reduced mitochondrial crista, and mitochondrial fragments. They also displayed decreased mitochondrial membrane potential, antioxidative ability, tricarboxylic acid cycle activity and energy production. At the same time, apoptosis and ferroptosis were increased. In addition, UMSC-P9, relative to UMSC-P4, showed undegraded materials in their lysosomes, the enhancement in lysosomal membrane permeability, the reduction in autophagy and phagocytosis. Moreover, transcriptome sequence analysis also revealed a reduction of cell function, metabolism, mitochondrial biogenesis, DNA replication and repair, and an increase of gene expression related to cell senescence, cancer, diseases, and infection in UMSC-P9.
CONCLUSION: This study indicates that in vitro long-time culturing of MSCs can cause mitochondrial and lysosomal dysfunction, probably contributing to the decline of cell activity and cell aging. Therefore, the morphology and function of mitochondria and lysosomes can be regarded as two important parameters to monitor cell viability, and they can also serve as two important indicators for optimizing in vitro culture conditions.

Keywords:  Cell activity; Human umbilical cord mesenchymal stem cells; Lysosomal dysfunction; Mitochondrial dysfunction

DOI:  https://doi.org/10.1186/s13287-022-03107-4
PLoS Pathog. 2022 Aug 15. 18(8): e1010543

Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.

Takayoshi Shirasaki, Hui Feng, Helen M E Duyvesteyn, William G Fusco, Kevin L McKnight, Ling Xie, Mark Boyce, Sathish Kumar, Rina Barouch-Bentov, Olga González-López, Ryan McNamara, Li Wang, Adriana Hertel-Wulff, Xian Chen, Shirit Einav, Joseph A Duncan, Maryna Kapustina, Elizabeth E Fry, David I Stuart, Stanley M Lemon.

Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.

DOI: https://doi.org/10.1371/journal.ppat.1010543
Elife. 2022 Aug 16. pii: e75250. [Epub ahead of print]11

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Rafael Romero-Becerra, Alfonso Mora, Elisa Manieri, Ivana Nikolic, Ayelén Melina Santamans, Valle Montalvo-Romeral, Francisco Miguel Cruz, Elena Rodríguez, Marta León, Luis Leiva-Vega, Laura Sanz, Víctor Bondía, David Filgueiras-Rama, Luis Jesús Jiménez-Borreguero, José Jalife, Barbara Gonzalez-Teran, Guadalupe Sabio.

  Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38β, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.

Keywords:  MKK3; MKK6; aging; cardiac hypertrophy; cell biology; mTOR; medicine; mouse; p38 MAPK

DOI:  https://doi.org/10.7554/eLife.75250
Elife. 2022 Aug 16. pii: e79830. [Epub ahead of print]11

Neuroinflammation in neuronopathic Gaucher disease: Role of microglia and NK cells, biomarkers, and response to substrate reduction therapy.

Chandra Sekhar Boddupalli, Shiny Nair, Glenn Belinsky, Joseph Gans, Erin Teeple, Tri-Hung Nguyen, Sameet Mehta, Lilu Guo, Martin L Kramer, Jiapeng Ruan, Honggge Wang, Matthew Davison, Dinesh Kumar, D J Vidyadhara, Bailin Zhang, Katherine Klinger, Pramod K Mistry.

   Background: Neuronopathic Gaucher disease (nGD) is a rare neurodegenerative disorder caused by biallelic mutations in GBA and buildup of glycosphingolipids in lysosomes. Neuronal injury and cell death are prominent pathological features; however, the role of GBA in individual cell types and involvement of microglia, blood-derived macrophages, and immune infiltrates in nGD pathophysiology remains enigmatic.
Methods: Here, using single-cell resolution of mouse nGD brains, lipidomics, and newly generated biomarkers, we found induction of neuroinflammation pathways involving microglia, NK cells, astrocytes, and neurons.
Results: Targeted rescue of Gba in microglia and neurons, respectively, in Gba-deficient, nGD mice reversed the buildup of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph), concomitant with amelioration of neuroinflammation, reduced serum neurofilament light chain (Nf-L), and improved survival. Serum GlcSph concentration was correlated with serum Nf-L and ApoE in nGD mouse models as well as in GD patients. Gba rescue in microglia/macrophage compartment prolonged survival, which was further enhanced upon treatment with brain-permeant inhibitor of glucosylceramide synthase, effects mediated via improved glycosphingolipid homeostasis, and reversal of neuroinflammation involving activation of microglia, brain macrophages, and NK cells.
Conclusions: Together, our study delineates individual cellular effects of Gba deficiency in nGD brains, highlighting the central role of neuroinflammation driven by microglia activation. Brain-permeant small-molecule inhibitor of glucosylceramide synthase reduced the accumulation of bioactive glycosphingolipids, concomitant with amelioration of neuroinflammation involving microglia, NK cells, astrocytes, and neurons. Our findings advance nGD disease biology whilst identifying compelling biomarkers of nGD to improve patient management, enrich clinical trials, and illuminate therapeutic targets.
Funding: Research grant from Sanofi; other support includes R01NS110354, Yale Liver Center P30DK034989, pilot project grant.

Keywords:  GBA; Gaucher disease; NK cells; human; lipids; medicine; microglia; mouse; neuroinflammation

DOI:  https://doi.org/10.7554/eLife.79830
J Cell Sci. 2022 Aug 15. pii: jcs260112. [Epub ahead of print]135(16):

Rerouting trafficking circuits through posttranslational SNARE modifications.

Harry Warner, Shweta Mahajan, Geert van den Bogaart.

  Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are membrane-associated trafficking proteins that confer identity to lipid membranes and facilitate membrane fusion. These functions are achieved through the complexing of Q-SNAREs with a specific cognate target R-SNARE, leading to the fusion of their associated membranes. These SNARE complexes then dissociate so that the Q-SNAREs and R-SNAREs can repeat this cycle. Whilst the basic function of SNAREs has been long appreciated, it is becoming increasingly clear that the cell can control the localisation and function of SNARE proteins through posttranslational modifications (PTMs), such as phosphorylation and ubiquitylation. Whilst numerous proteomic methods have shown that SNARE proteins are subject to these modifications, little is known about how these modifications regulate SNARE function. However, it is clear that these PTMs provide cells with an incredible functional plasticity; SNARE PTMs enable cells to respond to an ever-changing extracellular environment through the rerouting of membrane traffic. In this Review, we summarise key findings regarding SNARE regulation by PTMs and discuss how these modifications reprogramme membrane trafficking pathways.

Keywords:  Membrane traffic; Posttranslational modifications; SNARE proteins

DOI:  https://doi.org/10.1242/jcs.260112
Nucleic Acids Res. 2022 Aug 18. pii: gkac696. [Epub ahead of print]

Structural basis of 3'-end poly(A) RNA recognition by LARP1.

Guennadi Kozlov, Sandy Mattijssen, Jianning Jiang, Samuel Nyandwi, Tara Sprules, James R Iben, Steven L Coon, Sergei Gaidamakov, Anne M Noronha, Christopher J Wilds, Richard J Maraia, Kalle Gehring.

La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3'-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3' protection activity.

DOI: https://doi.org/10.1093/nar/gkac696
Mol Neurodegener. 2022 Aug 17. 17(1): 52

Transcriptome deregulation of peripheral monocytes and whole blood in GBA-related Parkinson's disease.

Giulietta Maria Riboldi, Ricardo A Vialle, Elisa Navarro, Evan Udine, Katia de Paiva Lopes, Jack Humphrey, Amanda Allan, Madison Parks, Brooklyn Henderson, Kelly Astudillo, Charalambos Argyrou, Maojuan Zhuang, Tamjeed Sikder, J Oriol Narcis, Shilpa Dilip Kumar, William Janssen, Allison Sowa, Giacomo P Comi, Alessio Di Fonzo, John F Crary, Steven J Frucht, Towfique Raj.

   BACKGROUND: Genetic mutations in beta-glucocerebrosidase (GBA) represent the major genetic risk factor for Parkinson's disease (PD). GBA participates in both the endo-lysosomal pathway and the immune response, two important mechanisms involved in the pathogenesis of PD. However, modifiers of GBA penetrance have not yet been fully elucidated.
METHODS: We characterized the transcriptomic profiles of circulating monocytes in a population of patients with PD and healthy controls (CTRL) with and without GBA variants (n = 23 PD/GBA, 13 CTRL/GBA, 56 PD, 66 CTRL) and whole blood (n = 616 PD, 362 CTRL, 127 PD/GBA, 165 CTRL/GBA). Differential expression analysis, pathway enrichment analysis, and outlier detection were performed. Ultrastructural characterization of isolated CD14+ monocytes in the four groups was also performed through electron microscopy.
RESULTS: We observed hundreds of differentially expressed genes and dysregulated pathways when comparing manifesting and non-manifesting GBA mutation carriers. Specifically, when compared to idiopathic PD, PD/GBA showed dysregulation in genes involved in alpha-synuclein degradation, aging and amyloid processing. Gene-based outlier analysis confirmed the involvement of lysosomal, membrane trafficking, and mitochondrial processing in manifesting compared to non-manifesting GBA-carriers, as also observed at the ultrastructural levels. Transcriptomic results were only partially replicated in an independent cohort of whole blood samples, suggesting cell-type specific changes.
CONCLUSIONS: Overall, our transcriptomic analysis of primary monocytes identified gene targets and biological processes that can help in understanding the pathogenic mechanisms associated with GBA mutations in the context of PD.

Keywords:  GBA; Monocytes; Parkinson’s disease; Transcriptomic analysis; beta-glucocerebrosidase

DOI:  https://doi.org/10.1186/s13024-022-00554-8
Elife. 2022 Aug 18. pii: e82041. [Epub ahead of print]11

Double NPY motifs at the N-terminus of the yeast t-SNARE Sso2 synergistically bind Sec3 to promote membrane fusion.

Maximilian Peer, Hua Yuan, Yubo Zhang, Katharina Korbula, Peter Novick, Gang Dong.

  Exocytosis is an active vesicle trafficking process by which eukaryotes secrete materials to the extracellular environment and insert membrane proteins into the plasma membrane. The final step of exocytosis in yeast involves the assembly of two t-SNAREs, Sso1/2 and Sec9, with the v-SNARE, Snc1/2, on secretory vesicles. The rate-limiting step in this process is the formation of a binary complex of the two t-SNAREs. Despite a previous report of acceleration of binary complex assembly by Sec3, it remains unknown how Sso2 is efficiently recruited to the vesicle-docking site marked by Sec3. Here we report a crystal structure of the pleckstrin homology (PH) domain of Sec3 in complex with a nearly full-length version of Sso2 lacking only its C-terminal transmembrane helix. The structure shows a previously uncharacterized binding site for Sec3 at the N-terminus of Sso2, consisting of two highly conserved triple residue motifs (NPY: Asn-Pro-Tyr). We further reveal that the two NPY motifs bind Sec3 synergistically, which together with the previously reported binding interface constitute dual-site interactions between Sso2 and Sec3 to drive the fusion of secretory vesicles at target sites on the plasma membrane.

Keywords:  S. cerevisiae; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.82041
Mol Biol Cell. 2022 Aug 17. mbcE22050177

The adaptor protein chaperone AAGAB stabilizes AP-4 complex subunits.

Rafael Mattera, Raffaella De Pace, Juan S Bonifacino.

Adaptor protein 4 (AP-4) is a heterotetrameric complex composed of ε, β4, μ4 and σ4 subunits that mediates export of a subset of transmembrane cargos, including autophagy protein 9A (ATG9A), from the trans-Golgi network (TGN). AP-4 has received particular attention in recent years because mutations in any of its subunits cause a complicated form of hereditary spastic paraplegia (HSP or SPG) referred to as "AP-4-deficiency syndrome." The identification of proteins that interact with AP-4 has shed light on the mechanisms of AP-4-dependent cargo sorting and distribution within the cell. However, the mechanisms by which the AP-4 complex itself is assembled have remained unknown. Herein, we report that the alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34) binds to and stabilizes the AP-4 ε-and σ4 subunits, thus promoting complex assembly. The importance of this binding is underscored by the observation that AAGAB-knockout cells exhibit reduced levels of AP-4 subunits and accumulation of ATG9A at the TGN like those in cells, mice, or patients with mutations in AP-4-subunit genes. These findings demonstrate that AP-4 assembly is not spontaneous but AAGAB-assisted, thus contributing to the understanding of an adaptor protein complex that is critically involved in development of the central nervous system.

DOI: https://doi.org/10.1091/mbc.E22-05-0177
Int J Biol Sci. 2022 ;18(13): 4869-4883

Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells.

Huiling Zhou, Qing Guan, Xuyang Hou, Lijun Liu, Li Zhou, Wei Li, Haidan Liu.

  Non-small cell lung cancer (NSCLC) is one of the deadliest cancers in the world. Metastasis is considered one of the leading causes of treatment failure and death in NSCLC patients. A crucial factor of promoting metastasis in epithelium-derived carcinoma has been considered as epithelial-mesenchymal transition (EMT). Rictor, one of the components of mTORC2, has been reportedly involved in EMT and metastasis of human malignancies. However, the regulatory mechanisms of Rictor, Rictor-mediated EMT and metastasis in cancers remain unknown. Our present study indicates that Rictor is highly expressed in human NSCLC cell lines and tissues and is regulated, at least partially, at the transcriptional level. Knockdown of Rictor expression causes phenotype alterations through EMT, which is accompanied by the impairment of migration and invasion ability in NSCLC cells. Additionally, we have cloned and identified the human Rictor core promoter for the first time and confirmed that transcription factor KLF4 directly binds to the Rictor promoter and transcriptionally upregulated Rictor expression. Knockdown of KLF4 results in Rictor's downregulation accompanied by a series of characteristic changes of mesenchymal-epithelial transition (MET) and significantly decreases migration, invasion as well as metastasis of NSCLC cells. Re-introducing Rictor in KLF4-knockdown NSCLC cells partially reverses the epithelial phenotype to the mesenchymal phenotype and attenuates the inhibition of cell migration and invasion caused by KLF4 knocking down. Knockdown of KLF4 prevents mTOR/Rictor interaction and metastasis of NSCLC in vivo. The understanding of the regulator upstream of Rictor may provide an opportunity for the development of new inhibitors and the rational design of combination regimens based on different metastasis-related molecular targets and finally prevents cancer metastasis.

Keywords:  EMT; KLF4; NSCLC; Rictor; metastasis

DOI:  https://doi.org/10.7150/ijbs.73548
J Cell Biol. 2022 Oct 03. pii: e202112138. [Epub ahead of print]221(10):

Eps15/Pan1p is a master regulator of the late stages of the endocytic pathway.

Mariko Enshoji, Yoshiko Miyano, Nao Yoshida, Makoto Nagano, Minami Watanabe, Mayumi Kunihiro, Daria E Siekhaus, Junko Y Toshima, Jiro Toshima.

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases-including actin nucleation promoting factors-and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.

DOI: https://doi.org/10.1083/jcb.202112138