bims-proteo 2023-06-11 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–06–11
28 papers selected by
Eric Chevet, INSERM

Rescue of proteotoxic stress and neurodegeneration by the Zn 2+ transporter ZIP7.
Mammalian ATG8 proteins maintain autophagosomal membrane integrity through ESCRTs.
The UBX domain in UBXD1 organizes ubiquitin binding at the C-terminus of the VCP/p97 AAA-ATPase.
The acyltransferase Gpc1 is both a target and an effector of the unfolded protein response (UPR) in Saccharomyces cerevisiae.
A pro-Fluorescent Ubiquitin-based Probe to Monitor Cysteine-based E3 Ligase Activity.
GPI-anchored Gas1 protein regulates cytosolic proteostasis in yeast.
Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s.
A cytosolic surveillance mechanism activates the mitochondrial UPR.
Valosin-containing protein (VCP/p97) is prognostically unfavorable in pediatric AML, and negatively correlates with unfolded protein response proteins IRE1 and GRP78: A report from the Children's Oncology Group.
The p97/VCP adapter UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.
mTORC1 inhibition impairs activation of the unfolded protein response and induces cell death during ER stress in cardiomyocytes.
PtdIns4P exchange at endoplasmic reticulum-autolysosome contacts is essential for autophagy and neuronal homeostasis.
Profiling of purified autophagic vesicle degradome in the maturing and aging brain.
Syntenin and CD63 Promote Exosome Biogenesis from the Plasma Membrane by Blocking Cargo Endocytosis.
Early and late chaperone intervention therapy boosts XBP1s and ADAM10, restores proteostasis, and rescues learning in Alzheimer's Disease mice.
Proximity Proteomics and Biochemical Analysis Reveal a Noncanonical Function for UFM1-Specific Protease 1 in the p62 Body Formation.
The GCN2/eIF2αK stress kinase regulates PP1 to ensure mitotic fidelity.
The Roles of Molecular Chaperones in Regulating Cell Metabolism.
Architecture and dynamics of a desmosome-endoplasmic reticulum complex.
Multitranscript analysis reveals an effect of 2-deoxy-d-glucose on gene expression linked to unfolded protein response and integrated stress response in primary human monocytes and monocyte-derived macrophages.
Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming.
Mycolactone A vs. B: Does localization or association explain isomer-specific toxicity?
Tisp40 prevents cardiac ischemia/reperfusion injury through the hexosamine biosynthetic pathway in male mice.
Triiodothyronine positively regulates endoplasmic reticulum-associated degradation (ERAD) and promotes androgenic signaling in androgen-dependent prostate cancer cells.
Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance.
Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.
Design of Cell-Permeable Inhibitors of Eukaryotic Translation Initiation Factor 4E (eIF4E) for Inhibiting Aberrant Cap-Dependent Translation in Cancer.
High-resolution structural-omics of human liver enzymes.

bioRxiv. 2023 May 22. pii: 2023.05.22.541645. [Epub ahead of print]

Rescue of proteotoxic stress and neurodegeneration by the Zn 2+ transporter ZIP7.

Xiaoran Guo, Morgan Mutch, Alba Yurani Torres, Maddalena Nano, Drew McDonald, Zijing Chen, Craig Montell, Wei Dai, Denise J Montell.

Proteotoxic stress drives numerous degenerative diseases. In response to misfolded proteins, cells adapt by activating the unfolded protein response (UPR), including endoplasmic reticulum-associated protein degradation (ERAD). However persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. From plants to humans, loss of the Zn 2+ transporter ZIP7 causes ER stress, however the mechanism is unknown. Here we show that ZIP7 enhances ERAD and that cytosolic Zn 2+ is limiting for deubiquitination of client proteins by the Rpn11 Zn 2+ metalloproteinase as they enter the proteasome in Drosophila and human cells. ZIP7 overexpression rescues defective vision caused by misfolded rhodopsin in Drosophila. Thus ZIP7 overexpression may prevent diseases caused by proteotoxic stress, and existing ZIP inhibitors may be effective against proteasome-dependent cancers.
One-Sentence Summary: Zn 2+ transport from the ER to the cytosol promotes deubiquitination and proteasomal degradation of misfolded proteins and prevents blindness in a fly neurodegeneration model.

DOI: https://doi.org/10.1101/2023.05.22.541645
EMBO J. 2023 Jun 05. e112845

Mammalian ATG8 proteins maintain autophagosomal membrane integrity through ESCRTs.

Ruheena Javed, Ashish Jain, Thabata Duque, Emily Hendrix, Masroor Ahmad Paddar, Sajjad Khan, Aurore Claude-Taupin, Jingyue Jia, Lee Allers, Fulong Wang, Michal Mudd, Graham Timmins, Keith Lidke, Tor Erik Rusten, Prithvi Reddy Akepati, Yi He, Fulvio Reggiori, Eeva-Liisa Eskelinen, Vojo Deretic.

  The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT-I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes.

Keywords:  ATG8; ESCRT; LC3; amphisome; autophagy

DOI:  https://doi.org/10.15252/embj.2022112845
Nat Commun. 2023 Jun 05. 14(1): 3258

The UBX domain in UBXD1 organizes ubiquitin binding at the C-terminus of the VCP/p97 AAA-ATPase.

Mike Blueggel, Alexander Kroening, Matthias Kracht, Johannes van den Boom, Matthias Dabisch, Anna Goehring, Farnusch Kaschani, Markus Kaiser, Peter Bayer, Hemmo Meyer, Christine Beuck.

The AAA+ ATPase p97/VCP together with different sets of substrate-delivery adapters and accessory cofactor proteins unfolds ubiquitinated substrates to facilitate degradation by the proteasome. The UBXD1 cofactor is connected to p97-associated multisystem proteinopathy but its biochemical function and structural organization on p97 has remained largely elusive. Using a combination of crosslinking mass spectrometry and biochemical assays, we identify an extended UBX (eUBX) module in UBXD1 related to a lariat in another cofactor, ASPL. Of note, the UBXD1-eUBX intramolecularly associates with the PUB domain in UBXD1 close to the substrate exit pore of p97. The UBXD1 PUB domain can also bind the proteasomal shuttling factor HR23b via its UBL domain. We further show that the eUBX domain has ubiquitin binding activity and that UBXD1 associates with an active p97-adapter complex during substrate unfolding. Our findings suggest that the UBXD1-eUBX module receives unfolded ubiquitinated substrates after they exit the p97 channel and before hand-over to the proteasome. The interplay of full-length UBXD1 and HR23b and their function in the context of an active p97:UBXD1 unfolding complex remains to be studied in future work.

DOI: https://doi.org/10.1038/s41467-023-38604-4
J Biol Chem. 2023 Jun 01. pii: S0021-9258(23)01912-9. [Epub ahead of print] 104884

The acyltransferase Gpc1 is both a target and an effector of the unfolded protein response (UPR) in Saccharomyces cerevisiae.

Victoria Lee Hrach, William R King, Laura D Nelson, Shane Conklin, John A Pollock, Jana Patton-Vogt.

  The unfolded protein response (UPR) is sensitive to proteotoxic and membrane bilayer stress, both of which are sensed by the ER protein Ire1. When activated, Ire1 splices HAC1 mRNA, producing a transcription factor that targets genes involved in proteostasis and lipid metabolism, among others. The major membrane lipid phosphatidylcholine (PC) is subject to phospholipase-mediated deacylation, producing glycerophosphocholine (GPC), followed by reacylation of GPC through the PC deacylation/reacylation pathway (PC-DRP). The reacylation events occur via a two-step process catalyzed first by the GPC acyltransferase Gpc1, followed by acylation of the lyso-PC molecule by Ale1. However, whether Gpc1 is critical for ER bilayer homeostasis is unclear. Using an improved method for C14-choline-GPC radiolabeling, we first show that loss of Gpc1 results in abrogation of PC synthesis through PC-DRP and that Gpc1 colocalizes with the ER. We then probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR-inducing compounds tunicamycin, DTT, and canavanine results in a Hac1-dependent increase in GPC1 message. Further, cells lacking Gpc1 exhibit increased sensitivity to those proteotoxic stressors. Inositol limitation, known to induce the UPR via bilayer stress, also induces GPC1 expression. Finally, we show that loss of GPC1 induces the UPR. A gpc1Δ mutant displays upregulation of the UPR in strains expressing a mutant form of Ire1 that is unresponsive to unfolded proteins, indicating that bilayer stress is responsible for the observed upregulation. Collectively, our data indicate an important role for Gpc1 in yeast ER bilayer homeostasis.

Keywords:  Gpc1; Ire1; acyltransferase; glycerophosphocholine; lipid remodeling; lysophosphatidylcholine; membrane bilayer stress; phosphatidylcholine; phospholipid metabolism; unfolded protein response; yeast

DOI:  https://doi.org/10.1016/j.jbc.2023.104884
Angew Chem Int Ed Engl. 2023 Jun 05. e202303319

A pro-Fluorescent Ubiquitin-based Probe to Monitor Cysteine-based E3 Ligase Activity.

David Pérez Berrocal, Thimmalapura Vishwanatha, Daniel Horn Ghetko, Josephine Bosch, Laura Hehl, Sebastian Kostrhon, Mohit Misra, Ivan Dikic, Paul Geurink, Hans van Dam, Brenda Schulman, Monique Priscilla Catharina Mulder.

  Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.

Keywords:  Fluorescent probes; Ligases; Profiling transthiolation activity; biological activity; drug discovery

DOI:  https://doi.org/10.1002/anie.202303319
bioRxiv. 2023 May 26. pii: 2023.05.26.542479. [Epub ahead of print]

GPI-anchored Gas1 protein regulates cytosolic proteostasis in yeast.

Yuhao Wang, Linhao Ruan, Rong Li.

Decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation via 'mitochondrial as guardian in cytosol' (MAGIC) pathway. Here we report an unexpected role of yeast Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of Gas1 inhibits MAGIC but elevates polyubiquitination and UPS-mediated protein degradation. Interestingly, we found that Gas1 exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins via MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1 E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.

DOI: https://doi.org/10.1101/2023.05.26.542479
Elife. 2023 Jun 08. pii: e89185. [Epub ahead of print]12

Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s.

Wenxin Zhang, Taki Nishimura, Deepanshi Gahlot, Chieko Saito, Colin Davis, Harold B J Jefferies, Anne Schreiber, Lipi Thukral, Sharon A Tooze.

  Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis-membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.

Keywords:  biochemistry; cell biology; chemical biology; none

DOI:  https://doi.org/10.7554/eLife.89185
Nature. 2023 Jun 07.

A cytosolic surveillance mechanism activates the mitochondrial UPR.

F X Reymond Sutandy, Ines Gößner, Georg Tascher, Christian Münch.

The mitochondrial unfolded protein response (UPRmt) is essential to safeguard mitochondria from proteotoxic damage by activating a dedicated transcriptional response in the nucleus to restore proteostasis1,2. Yet, it remains unclear how the information on mitochondria misfolding stress (MMS) is signalled to the nucleus as part of the human UPRmt (refs. 3,4). Here, we show that UPRmt signalling is driven by the release of two individual signals in the cytosol-mitochondrial reactive oxygen species (mtROS) and accumulation of mitochondrial protein precursors in the cytosol (c-mtProt). Combining proteomics and genetic approaches, we identified that MMS causes the release of mtROS into the cytosol. In parallel, MMS leads to mitochondrial protein import defects causing c-mtProt accumulation. Both signals integrate to activate the UPRmt; released mtROS oxidize the cytosolic HSP40 protein DNAJA1, which leads to enhanced recruitment of cytosolic HSP70 to c-mtProt. Consequently, HSP70 releases HSF1, which translocates to the nucleus and activates transcription of UPRmt genes. Together, we identify a highly controlled cytosolic surveillance mechanism that integrates independent mitochondrial stress signals to initiate the UPRmt. These observations reveal a link between mitochondrial and cytosolic proteostasis and provide molecular insight into UPRmt signalling in human cells.

DOI: https://doi.org/10.1038/s41586-023-06142-0
Proteomics Clin Appl. 2023 Jun 07. e2200109

Valosin-containing protein (VCP/p97) is prognostically unfavorable in pediatric AML, and negatively correlates with unfolded protein response proteins IRE1 and GRP78: A report from the Children's Oncology Group.

Fieke W Hoff, Yihua Qiu, Brandon D Brown, Robert B Gerbing, Amanda R Leonti, Rhonda E Ries, Alan S Gamis, Richard Aplenc, Edward Anders Kolb, Todd A Alonzo, Soheil Meshinchi, Gaye N Jenkins, Terzah M Horton, Steven M Kornblau.

   PURPOSE: The endoplasmic reticulum (ER) is the major site of protein synthesis and folding in the cell. ER-associated degradation (ERAD) and unfolded protein response (UPR) are the main mechanisms of ER-mediated cell stress adaptation. Targeting the cell stress response is a promising therapeutic approach in acute myeloid leukemia (AML).
EXPERIMENTAL DESIGN: Protein expression levels of valosin-containing protein (VCP), a chief element of ERAD, were measured in peripheral blood samples from in 483 pediatric AML patients using reverse phase protein array methodology. Patients participated in the Children's Oncology Group AAML1031 phase 3 clinical trial that randomized patients to standard chemotherapy (cytarabine (Ara-C), daunorubicin, and etoposide [ADE]) versus ADE plus bortezomib (ADE+BTZ).
RESULTS: Low-VCP expression was significantly associated with favorable 5-year overall survival (OS) rate compared to middle-high-VCP expression (81% versus 63%, p < 0.001), independent of additional bortezomib treatment. Multivariable Cox regression analysis identified VCP as independent predictor of clinical outcome. UPR proteins IRE1 and GRP78 had significant negative correlation with VCP. Five-year OS in patients characterized by low-VCP, moderately high-IRE1 and high-GRP78 improved after treatment with ADE+BTZ versus ADE (66% versus 88%, p = 0.026).
CONCLUSION AND CLINICAL RELEVANCE: Our findings suggest the potential of the protein VCP as biomarker in prognostication prediction in pediatric AML.

Keywords:  AML; ERAD; GRP78; IRE1; RPPA; UPR; VCP ; leukemia; pediatric

DOI:  https://doi.org/10.1002/prca.202200109
bioRxiv. 2023 May 15. pii: 2023.05.15.540864. [Epub ahead of print]

The p97/VCP adapter UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.

Julian R Braxton, Chad R Altobelli, Maxwell R Tucker, Eric Tse, Aye C Thwin, Michelle R Arkin, Daniel R Southworth.

p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adapter localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. We identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact p97:UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX, and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second AAA+ domain. Together these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis, and comparisons to other adapters further reveal how adapters containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.

DOI: https://doi.org/10.1101/2023.05.15.540864
Am J Physiol Heart Circ Physiol. 2023 Jun 09.

mTORC1 inhibition impairs activation of the unfolded protein response and induces cell death during ER stress in cardiomyocytes.

Christoph Hofmann, Zoe Löwenthal, Marjan Aghajani, Randal J Kaufman, Hugo A Katus, Norbert Frey, Christopher C Glembotski, Mirko Völkers, Shirin Doroudgar.

  The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of protein synthesis that senses and responds to a variety of stimuli to coordinate cellular metabolism with environmental conditions. To ensure that protein synthesis is inhibited during unfavorable conditions, translation is directly coupled to the sensing of cellular protein homeostasis. Thus, translation is attenuated during endoplasmic reticulum (ER) stress by direct inhibition of the mTORC1 pathway. However, residual mTORC1 activity is maintained during prolonged ER stress which is thought to be involved in translational reprogramming and adaption to ER stress. By analyzing the dynamics of mTORC1 regulation during ER stress, we unexpectedly found that mTORC1 is transiently activated in cardiomyocytes within minutes at the onset of ER stress before being inhibited during chronic ER stress. This dynamic regulation of mTORC1 appears to be mediated, at least in part, by ATF6, as its activation was sufficient to induce the biphasic control of mTORC1. We further showed that protein synthesis remains dependent on mTORC1 throughout the ER stress response and that mTORC1 activity is essential for posttranscriptional induction of several unfolded protein response elements. Pharmacological inhibition of mTORC1 increased cell death during ER stress, indicating that the mTORC1 pathway serves adaptive functions during ER stress in cardiomyocytes potentially by controlling the expression of the protective unfolded protein response.

Keywords:  ATF6; ER stress; cardiomyocytes; cell death; mTORC1

DOI:  https://doi.org/10.1152/ajpheart.00682.2022
Autophagy. 2023 Jun 08.

PtdIns4P exchange at endoplasmic reticulum-autolysosome contacts is essential for autophagy and neuronal homeostasis.

Hao Liu, Wenxia Shao, Wei Liu, Weina Shang, Jun-Ping Liu, Liquan Wang, Chao Tong.

  Inter-organelle contacts enable crosstalk among organelles, facilitating the exchange of materials and coordination of cellular events. In this study, we demonstrated that, upon starvation, autolysosomes recruit Pi4KIIα (Phosphatidylinositol 4-kinase II α) to generate phosphatidylinositol-4-phosphate (PtdIns4P) on their surface and establish endoplasmic reticulum (ER)-autolysosome contacts through PtdIns4P binding proteins Osbp (Oxysterol binding protein) and cert (ceramide transfer protein). We found that the Sac1 (Sac1 phosphatase), Osbp, and cert proteins are required for the reduction of PtdIns4P on autolysosomes. Loss of any of these proteins leads to defective macroautophagy/autophagy and neurodegeneration. Osbp, cert, and Sac1 are required for ER-Golgi contacts in fed cells. Our data establishes a new mode of organelle contact formation-the ER-Golgi contact machinery can be reused by ER-autolysosome contacts by re-locating PtdIns4P from the Golgi apparatus to autolysosomes when faced with starvation.

Keywords:  Drosophila; Golgi apparatus; PtdIns4P; endoplasmic reticulum-autolysosome contacts

DOI:  https://doi.org/10.1080/15548627.2023.2222556
Neuron. 2023 May 26. pii: S0896-6273(23)00384-7. [Epub ahead of print]

Profiling of purified autophagic vesicle degradome in the maturing and aging brain.

Emmanouela Kallergi, Devanarayanan Siva Sankar, Alessandro Matera, Angeliki Kolaxi, Rosa Chiara Paolicelli, Joern Dengjel, Vassiliki Nikoletopoulou.

  Autophagy disorders prominently affect the brain, entailing neurodevelopmental and neurodegenerative phenotypes in adolescence or aging, respectively. Synaptic and behavioral deficits are largely recapitulated in mouse models with ablation of autophagy genes in brain cells. Yet, the nature and temporal dynamics of brain autophagic substrates remain insufficiently characterized. Here, we immunopurified LC3-positive autophagic vesicles (LC3-pAVs) from the mouse brain and proteomically profiled their content. Moreover, we characterized the LC3-pAV content that accumulates after macroautophagy impairment, validating a brain autophagic degradome. We reveal selective pathways for aggrephagy, mitophagy, and ER-phagy via selective autophagy receptors, and the turnover of numerous synaptic substrates, under basal conditions. To gain insight into the temporal dynamics of autophagic protein turnover, we quantitatively compared adolescent, adult, and aged brains, revealing critical periods of enhanced mitophagy or degradation of synaptic substrates. Overall, this resource unbiasedly characterizes the contribution of autophagy to proteostasis in the maturing, adult, and aged brain.

Keywords:  ER-phagy; aggrephagy; autophagic vesicles; brain; degradome; mitophagy; proteomic profiling; synapse

DOI:  https://doi.org/10.1016/j.neuron.2023.05.011
bioRxiv. 2023 May 26. pii: 2023.05.26.542409. [Epub ahead of print]

Syntenin and CD63 Promote Exosome Biogenesis from the Plasma Membrane by Blocking Cargo Endocytosis.

Yiwei Ai, Chenxu Guo, Marta Garcia-Contreras, Laura S Sánchez B, Andras Saftics, Oluwapelumi Shodubi, Shankar Raghunandan, Junhao Xu, Shang Jui Tsai, Yi Dong, Rong Li, Tijana Jovanovic-Talisman, Stephen J Gould.

Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the endosomal biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosome-localized CD63. We find instead that syntenin blocks CD63 endocytosis, allowing CD63 to accumulate at the plasma membrane, thereby increasing its direct budding from the plasma membrane. Consistent with this model, we show that specific and general inhibitors of endocytosis induce the exosomal secretion of CD63 and that endocytosis signals inhibit the vesicular secretion of exosome cargo proteins. Furthermore, we show that CD63 is itself a competitive inhibitor of AP-2-mediated endocytosis, driving the plasma membrane accumulation and exosomal secretion of itself and other lysosome membrane proteins. Our results support the hypothesis that highly-enriched exosome cargo proteins bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, and that syntenin and CD63 regulate the loading of lysosomal proteins into exosomes by an Alix-independent modulation of endocytosis.

DOI: https://doi.org/10.1101/2023.05.26.542409
bioRxiv. 2023 May 24. pii: 2023.05.23.541973. [Epub ahead of print]

Early and late chaperone intervention therapy boosts XBP1s and ADAM10, restores proteostasis, and rescues learning in Alzheimer's Disease mice.

Jennifer M Hafycz, Ewa Strus, Nirinjini N Naidoo.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder that is pervasive among the aging population. Two distinct phenotypes of AD are deficits in cognition and proteostasis, including chronic activation of the unfolded protein response (UPR) and aberrant Aβ production. It is unknown if restoring proteostasis by reducing chronic and aberrant UPR activation in AD can improve pathology and cognition. Here, we present data using an APP knock-in mouse model of AD and several protein chaperone supplementation paradigms, including a late-stage intervention. We show that supplementing protein chaperones systemically and locally in the hippocampus reduces PERK signaling and increases XBP1s, which is associated with increased ADAM10 and decreased Aβ42. Importantly, chaperone treatment improves cognition which is correlated with increased CREB phosphorylation and BDNF. Together, this data suggests that chaperone treatment restores proteostasis in a mouse model of AD and that this restoration is associated with improved cognition and reduced pathology.
One-sentence summary: Chaperone therapy in a mouse model of Alzheimer's disease improves cognition by reducing chronic UPR activity.

DOI: https://doi.org/10.1101/2023.05.23.541973
J Proteome Res. 2023 Jun 07.

Proximity Proteomics and Biochemical Analysis Reveal a Noncanonical Function for UFM1-Specific Protease 1 in the p62 Body Formation.

Xiaohui Wang, Lindong Cao, Honglv Jiang, Liang Zhou, Zhanhong Hu, Guoqiang Xu.

  Protein aggregates play crucial roles in the development of neurodegenerative diseases and p62 is one of the key proteins regulating the formation of protein aggregates. Recently, it has been discovered that depletion of several key enzymes including UFM1-activating enzyme UBA5, UFM1-conjugating enzyme UFC1, UFM1-protein ligase UFL1, and UFM1-specific protease UfSP2 in the UFM1-conjugation system induces p62 accumulation to form p62 bodies in the cytosol. However, it is unknown whether UfSP1 participates in the formation of p62 bodies and whether its enzymatic activity is required for this process. Here, the proximity labeling technique and quantitative proteomics identify SQSTM1/p62 as a UfSP1-interacting protein. Coimmunoprecipitation reveals that p62 indeed interacts with UfSP1 and the immunofluorescence experiment discloses that UfSP1 colocalizes with p62 and promotes the formation of p62-mediated protein aggregates. Mechanistic studies unveil that UfSP1 binds to the ubiquitin-associated domain of p62 and promotes the interaction between p62 and ubiquitinated proteins, thereby increasing the formation of p62 bodies. Interestingly, we further demonstrate that both the catalytic active and inactive UfSP1 promote the formation of p62 bodies through the same mechanism. Taken together, this work discovers that UfSP1 exhibits a noncanonical function independent of its protease activity in the p62 body formation.

Keywords:  TurboID; UFM1; UfSP1; noncanonical function; p62; p62 bodies; quantitative proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.3c00107
EMBO Rep. 2023 Jun 09. e56100

The GCN2/eIF2αK stress kinase regulates PP1 to ensure mitotic fidelity.

Vilte Stonyte, Maria Mastrangelopoulou, Romy Timmer, Lilian Lindbergsengen, Marina Vietri, Coen Campsteijn, Beáta Grallert.

  GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2-dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during early mitosis. These findings highlight a druggable PP1 inhibitor and open new avenues of research on the therapeutic potential of GCN2 inhibitors.

Keywords:  Aurora A; GCN2; PP1; chromosome alignment; mitosis

DOI:  https://doi.org/10.15252/embr.202256100
FEBS Lett. 2023 Jun 07.

The Roles of Molecular Chaperones in Regulating Cell Metabolism.

Matthew J Binder, Anthony M Pedley.

  Fluctuations in nutrient and biomass availability, often as a result of disease, impart metabolic challenges that must be overcome in order to sustain cell survival and promote proliferation. Cells adapt to these environmental changes and stresses by adjusting their metabolic networks through a series of regulatory mechanisms. Our understanding of these rewiring events has largely been focused on those genetic transformations that alter protein expression and the biochemical mechanisms that change protein behavior, such as post-translational modifications and metabolite-based allosteric modulators. Mounting evidence suggests that a class of proteome surveillance proteins called molecular chaperones also can influence metabolic processes. Here, we summarize several ways the Hsp90 and Hsp70 chaperone families act on human metabolic enzymes and their supramolecular assemblies to change enzymatic activities and metabolite flux. We further highlight how these chaperones can assist in the translocation and degradation of metabolic enzymes. Collectively, these studies provide a new view for how metabolic processes are regulated to meet cellular demand and inspire new avenues for therapeutic intervention.

Keywords:  cellular metabolism; metabolons; molecular chaperones; protein degradation; protein folding; supramolecular complexes

DOI:  https://doi.org/10.1002/1873-3468.14682
Nat Cell Biol. 2023 Jun 08.

Architecture and dynamics of a desmosome-endoplasmic reticulum complex.

COSEM Project Team

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

DOI: https://doi.org/10.1038/s41556-023-01154-4
Biochim Biophys Acta Gen Subj. 2023 Jun 06. pii: S0304-4165(23)00095-8. [Epub ahead of print] 130397

Multitranscript analysis reveals an effect of 2-deoxy-d-glucose on gene expression linked to unfolded protein response and integrated stress response in primary human monocytes and monocyte-derived macrophages.

Y S Tamayo-Molina, Paula A Velilla, Lady Johana Hernández-Sarmiento, Silvio Urcuqui-Inchima.

   BACKGROUND: Glycolytic inhibitor 2-deoxy-d-glucose (2-DG) binds to hexokinase in a non-competitive manner and phosphoglucose isomerase in a competitive manner, blocking the initial steps of the glycolytic pathway. Although 2-DG stimulates endoplasmic reticulum (ER) stress, activating the unfolded protein response to restore protein homeostasis, it is unclear which ER stress-related genes are modulated in response to 2-DG treatment in human primary cells. Here, we aimed to determine whether the treatment of monocytes and monocyte-derived macrophages (MDMs) with 2-DG leads to a transcriptional profile specific to ER stress.
METHODS: We performed bioinformatics analysis to identify differentially expressed genes (DEGs) in previously reported RNA-seq datasets of 2-DG treated cells. RT-qPCR was performed to verify the sequencing data on cultured MDMs.
RESULTS: A total of 95 common DEGs were found by transcriptional analysis of monocytes and MDMs treated with 2-DG. Among these, 74 were up-regulated and 21 were down-regulated. Multitranscript analysis showed that DEGs are linked to integrated stress response (GRP78/BiP, PERK, ATF4, CHOP, GADD34, IRE1α, XBP1, SESN2, ASNS, PHGDH), hexosamine biosynthetic pathway (GFAT1, GNA1, PGM3, UAP1), and mannose metabolism (GMPPA and GMPPB).
CONCLUSIONS: Results reveal that 2-DG triggers a gene expression program that might be involved in restoring protein homeostasis in primary cells.
GENERAL SIGNIFICANCE: 2-DG is known to inhibit glycolysis and induce ER stress; however, its effect on gene expression in primary cells is not well understood. This work shows that 2-DG is a stress inducer shifting the metabolic state of monocytes and macrophages.

Keywords:  Endoplasmic reticulum stress; Human primary cells; Integrated stress response; Protein glycosylation; Unfolded protein response

DOI:  https://doi.org/10.1016/j.bbagen.2023.130397
Commun Biol. 2023 06 03. 6(1): 602

Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming.

Navin Ray, Seung Jun Park, Hoyung Jung, Juil Kim, Tamas Korcsmaros, Yuseok Moon.

The integrated stress response (ISR) plays a pivotal role in the cellular stress response, primarily through global translational arrest and the upregulation of cellular adaptation-linked molecules. Growth differentiation factor 15 (Gdf15) is a potent stress-responsive biomarker of clinical inflammatory and metabolic distress in various types of diseases. Herein, we assess whether ISR-driven cellular stress contributes to pathophysiological outcomes by modulating Gdf15. Clinical transcriptome analysis demonstrates that PKR is positively associated with Gdf15 expression in patients with renal injury. Gdf15 expression is dependent on protein kinase R (PKR)-linked ISR during acute renointestinal distress in mice and genetic ablation of Gdf15 aggravates chemical-induced lesions in renal tissues and the gut barrier. An in-depth evaluation of the gut microbiota indicates that Gdf15 is associated with the abundance of mucin metabolism-linked bacteria and their enzymes. Moreover, stress-responsive Gdf15 facilitates mucin production and cellular survival via the reorganization of the autophagy regulatory network. Collectively, ISR-activated Gdf15 counteracts pathological processes via the protective reprogramming of the autophagic network and microbial community, thereby providing robust predictive biomarkers and interventions against renointestinal distress.

DOI: https://doi.org/10.1038/s42003-023-04965-1
bioRxiv. 2023 May 22. pii: 2023.05.19.541532. [Epub ahead of print]

Mycolactone A vs. B: Does localization or association explain isomer-specific toxicity?

John D M Nguyen, Gabriel C A da Hora, Jessica M J Swanson.

Mycolactone is an exotoxin produced by Mycobacterium ulcerans that causes the neglected tropical skin disease Buruli ulcer. This toxin inhibits the Sec61 translocon in the endoplasmic reticulum (ER), preventing the host cell from producing many secretory and transmembrane proteins, resulting in cytotoxic and immunomodulatory effects. Interestingly, only one of the two dominant isoforms of mycolactone is cytotoxic. Here, we investigate the origin of this specificity by performing extensive molecular dynamics (MD) simulations with enhanced free energy sampling to query the association trends of the two isoforms with both the Sec61 translocon and the ER membrane, which serves as a toxin reservoir prior to association. Our results suggest that mycolactone B (the cytotoxic isoform) has a stronger association with the ER membrane than mycolactone A due to more favorable interactions with membrane lipids and water molecules. This could increase the reservoir of toxin proximal to the Sec61 translocon. Isomer B also interacts more closely with the lumenal and lateral gates of the translocon, the dynamics of which are essential for protein translocation. These interactions induce a more closed conformation, which has been suggested to block signal peptide insertion and subsequent protein translocation. Collectively, these findings suggest that isomer B's unique cytotoxicity is a consequence of both increased localization to the ER membrane and channel-locking association with the Sec61 translocon, facets that could be targeted in the development of Buruli Ulcer diagnostics and Sec61-targeted therapeutics.

DOI: https://doi.org/10.1101/2023.05.19.541532
Nat Commun. 2023 Jun 08. 14(1): 3383

Tisp40 prevents cardiac ischemia/reperfusion injury through the hexosamine biosynthetic pathway in male mice.

Xin Zhang, Can Hu, Zhen-Guo Ma, Min Hu, Xiao-Pin Yuan, Yu-Pei Yuan, Sha-Sha Wang, Chun-Yan Kong, Teng Teng, Qi-Zhu Tang.

The hexosamine biosynthetic pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to facilitate O-linked GlcNAc (O-GlcNAc) protein modifications, and subsequently enhance cell survival under lethal stresses. Transcript induced in spermiogenesis 40 (Tisp40) is an endoplasmic reticulum membrane-resident transcription factor and plays critical roles in cell homeostasis. Here, we show that Tisp40 expression, cleavage and nuclear accumulation are increased by cardiac ischemia/reperfusion (I/R) injury. Global Tisp40 deficiency exacerbates, whereas cardiomyocyte-restricted Tisp40 overexpression ameliorates I/R-induced oxidative stress, apoptosis and acute cardiac injury, and modulates cardiac remodeling and dysfunction following long-term observations in male mice. In addition, overexpression of nuclear Tisp40 is sufficient to attenuate cardiac I/R injury in vivo and in vitro. Mechanistic studies indicate that Tisp40 directly binds to a conserved unfolded protein response element (UPRE) of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) promoter, and subsequently potentiates HBP flux and O-GlcNAc protein modifications. Moreover, we find that I/R-induced upregulation, cleavage and nuclear accumulation of Tisp40 in the heart are mediated by endoplasmic reticulum stress. Our findings identify Tisp40 as a cardiomyocyte-enriched UPR-associated transcription factor, and targeting Tisp40 may develop effective approaches to mitigate cardiac I/R injury.

DOI: https://doi.org/10.1038/s41467-023-39159-0
Cell Signal. 2023 Jun 02. pii: S0898-6568(23)00159-6. [Epub ahead of print] 110745

Triiodothyronine positively regulates endoplasmic reticulum-associated degradation (ERAD) and promotes androgenic signaling in androgen-dependent prostate cancer cells.

Yalcin Erzurumlu, Muhammed Tilahun Muhammed.

  Thyroid hormones (THs) play crucial roles in numerous physiological processes of nearly all mammalian tissues, including differentiation and metabolism. Deterioration of TH signaling has been associated with several pathologies, including cancer. The effect of highly active triiodothyronine (T3) has been investigated in many in vivo and in vitro cancer models. However, the role of T3 on cancerous prostate tissue is controversial. Recent studies have focused on the characterization of the supportive roles of the endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) signaling in prostate cancer (PCa) and investigating new hormonal regulation patterns, including estrogen, progesterone and 1,25(OH)2D3. Additionally, androgenic signaling controlled by androgens, which are critical in PCa progression, has been shown to be regulated by other steroid hormones. While the effects of T3 on ERAD and UPR are unknown today, the impact on androgenic signaling is still not understood in PCa. Therefore, we aimed to investigate the molecular action of T3 on the ERAD mechanism and UPR signaling in PCa cells and also extensively examined the effect of T3 on androgenic signaling. Our data strongly indicated that T3 tightly regulates ERAD and UPR signaling in androgen-dependent PCa cells. We also found that T3 stimulates androgenic signaling by upregulating AR mRNA and protein levels and enhancing its nuclear translocation. Additionally, advanced computational studies supported the ligand binding effect of T3 on AR protein. Our data suggest that targeting thyroidal signaling should be considered in therapeutic approaches to be developed for prostate malignancy in addition to other steroidal regulations.

Keywords:  Androgen receptor; Endoplasmic reticulum-associated degradation; Prostate cancer; Triiodothyronine; Unfolded protein response

DOI:  https://doi.org/10.1016/j.cellsig.2023.110745
bioRxiv. 2023 May 22. pii: 2023.05.17.541154. [Epub ahead of print]

Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance.

Neha Zutshi, Bhopal C Mohapatra, Pinaki Mondal, Wei An, Benjamin T Goetz, Shuo Wang, Sicong Li, Matthew D Storck, David F Mercer, Adrian R Black, Sarah P Thayer, Jennifer D Black, Chi Lin, Vimla Band, Hamid Band.

Among the signaling pathways that control the stem cell self-renewal and maintenance vs. acquisition of differentiated cell fates, those mediated by receptor tyrosine kinase (RTK) activation are well established as key players. CBL family ubiquitin ligases are negative regulators of RTKs but their physiological roles in regulating stem cell behaviors are unclear. While hematopoietic Cbl/Cblb knockout (KO) leads to a myeloproliferative disease due to expansion and reduced quiescence of hematopoietic stem cells, mammary epithelial KO led to stunted mammary gland development due to mammary stem cell depletion. Here, we examined the impact of inducible Cbl/Cblb double-KO (iDKO) selectively in the Lgr5-defined intestinal stem cell (ISC) compartment. Cbl/Cblb iDKO led to rapid loss of the Lgr5 Hi ISC pool with a concomitant transient expansion of the Lgr5 Lo transit amplifying population. LacZ reporter-based lineage tracing showed increased ISC commitment to differentiation, with propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro , Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single cell RNAseq analysis of organoids revealed Akt-mTOR pathway hyperactivation in iDKO ISCs and progeny cells, and pharmacological inhibition of the Akt-mTOR axis rescued the organoid maintenance and propagation defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

DOI: https://doi.org/10.1101/2023.05.17.541154
Nat Struct Mol Biol. 2023 Jun 08.

Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.

Nestor Miguel Castillo Duque de Estrada, Matthias Thoms, Dirk Flemming, Henrik M Hammaren, Robert Buschauer, Michael Ameismeier, Jochen Baßler, Martin Beck, Roland Beckmann, Ed Hurt.

The 5S ribonucleoprotein (RNP) is assembled from its three components (5S rRNA, Rpl5/uL18 and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2-p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-electron microscopy structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial nuclear import complex Syo1-uL18-uL5 and, upon further recruitment of the nucleolar factors Rpf2 and Rrs1, develops into the 5S RNP precursor that can assemble into the pre-ribosome. In addition, we elucidate the structure of another 5S RNP intermediate, carrying the human ubiquitin ligase Mdm2, which unravels how this enzyme can be sequestered from its target substrate p53. Our data provide molecular insight into how the 5S RNP can mediate between ribosome biogenesis and cell proliferation.

DOI: https://doi.org/10.1038/s41594-023-01006-7
bioRxiv. 2023 May 24. pii: 2023.05.23.541912. [Epub ahead of print]

Design of Cell-Permeable Inhibitors of Eukaryotic Translation Initiation Factor 4E (eIF4E) for Inhibiting Aberrant Cap-Dependent Translation in Cancer.

Emilio L Cárdenas, Rachel L O'Rourke, Arya Menon, Jennifer Meagher, Jeanne Stuckey, Amanda L Garner.

Eukaryotic translation initiation factor 4E (eIF4E) is an RNA-binding protein that binds to the m 7 GpppX-cap at the 5' terminus of coding mRNAs to initiate cap-dependent translation. While all cells require cap-dependent translation, cancer cells become addicted to enhanced translational capacity, driving the production of oncogenic proteins involved in proliferation, evasion of apoptosis, metastasis, and angiogenesis among other cancerous phenotypes. eIF4E is the rate-limiting translation factor and its activation has been shown to drive cancer initiation, progression, metastasis, and drug resistance. These findings have established eIF4E as a translational oncogene and promising, albeit challenging, anti-cancer therapeutic target. Although significant effort has been put forth towards inhibiting eIF4E, the design of cell-permeable, cap-competitive inhibitors remains a challenge. Herein, we describe our work towards solving this long-standing challenge. By employing an acyclic nucleoside phosphonate prodrug strategy, we report the synthesis of cell-permeable inhibitors of eIF4E binding to capped mRNA to inhibit cap-dependent translation.

DOI: https://doi.org/10.1101/2023.05.23.541912
Cell Rep. 2023 Jun 07. pii: S2211-1247(23)00620-4. [Epub ahead of print]42(6): 112609

High-resolution structural-omics of human liver enzymes.

Chih-Chia Su, Meinan Lyu, Zhemin Zhang, Masaru Miyagi, Wei Huang, Derek J Taylor, Edward W Yu.

  We applied raw human liver microsome lysate to a holey carbon grid and used cryo-electron microscopy (cryo-EM) to define its composition. From this sample we identified and simultaneously determined high-resolution structural information for ten unique human liver enzymes involved in diverse cellular processes. Notably, we determined the structure of the endoplasmic bifunctional protein H6PD, where the N- and C-terminal domains independently possess glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase enzymatic activity, respectively. We also obtained the structure of heterodimeric human GANAB, an ER glycoprotein quality-control machinery that contains a catalytic α subunit and a noncatalytic β subunit. In addition, we observed a decameric peroxidase, PRDX4, which directly contacts a disulfide isomerase-related protein, ERp46. Structural data suggest that several glycosylations, bound endogenous compounds, and ions associate with these human liver enzymes. These results highlight the importance of cryo-EM in facilitating the elucidation of human organ proteomics at the atomic level.

Keywords:  CP: Metabolism; CP: Molecular biology; aldehyde oxidase 1; carboxylesterase 1; glucosidase II; glutamate dehydrogenase 1; glycogen phosphorylase; hexose-6-phosphate dehydrogenase; microsomal triglyceride transfer protein complex; peroxiredoxin 4 – endoplasmic reticulum protein 46 complex; retinaldehyde dehydrogenase 1; structural-omics

DOI:  https://doi.org/10.1016/j.celrep.2023.112609