bims-proteo 2023-04-09 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–04–09
thirty papers selected by
Eric Chevet, INSERM

N-glycosylation mediated folding and quality control in serine proteases of the hepsin family.
The unfolded protein response transcription factor XBP1s ameliorates Alzheimer's disease by improving synaptic function and proteostasis.
The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia.
Reversible protein assemblies in the proteostasis network in health and disease.
The fifth element: a novel eIF2α kinase in microglia.
Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox.
Systemwide disassembly and assembly of SCF ubiquitin ligase complexes.
The dynamic role of endoplasmic reticulum stress in chronic liver disease.
The ubiquitin-like modifier FAT10 is degraded by the 20S proteasome in vitro but not in cellulo.
PERK prevents rhodopsin degradation during retinitis pigmentosa by inhibiting IRE1-induced autophagy.
The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia.
SENP3 and USP7 regulate Polycomb-rixosome interactions and silencing functions.
Contributions of Ccr4 and Gcn2 to the Translational Response of C. neoformans to Host-Relevant Stressors and Integrated Stress Response Induction.
Normal tissue homeostasis and impairment of selective inflammatory responses in dendritic cells deficient for ATF6α.
LC3-independent autophagy is vital to prevent TNF cytotoxicity.
Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis.
ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience.
PROTAC antibiotics: the time is now.
Biophysical and computational approaches to study ternary complexes: A 'cooperative relationship' to rationalize targeted protein degradation.
The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.
ERK1/2 inhibitors act as monovalent degraders inducing ubiquitylation and proteasome-dependent turnover of ERK2, but not ERK1.
HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development.
Emerging evidence for poxvirus-mediated unfolded protein response: Lumpy skin disease virus maintains self-replication by activating PERK and IRE1 signaling.
A lysosome membrane regeneration pathway depends on TBC1D15 and autophagic lysosomal reformation proteins.
Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control.
eIF2Bδ blocks the integrated stress response and maintains eIF2B activity and cancer metastasis by overexpression in breast cancer stem cells.
De novo design of modular peptide-binding proteins by superhelical matching.
Oncogenic CALR mutant C-terminus mediates dual binding to the thrombopoietin receptor triggering complex dimerization and activation.
Targeting USP2 regulation of VPRBP-mediated degradation of p53 and PD-L1 for cancer therapy.
Hepatocyte CD36 modulates UBQLN1-mediated proteasomal degradation of autophagic SNARE proteins contributing to septic liver injury.

FEBS J. 2023 Apr 04.

N-glycosylation mediated folding and quality control in serine proteases of the hepsin family.

John J M Bergeron, David Y Thomas.

  N-linked glycans are specifically attached to asparagine residues in a N-X-S/T motif of secretory pathway glycoproteins. N-glycosylation of newly synthesized glycoproteins directs their folding via the lectin chaperones calnexin and calreticulin that are associated with protein-folding enzymes and glycosidases of the endoplasmic reticulum (ER). Misfolded glycoproteins are retained in the ER by the same lectin chaperones. The work by Sun et al. (FEBS J 2023, 10.1111/febs.16757) in this issue focusses on hepsin, a serine protease on the surface of liver and other organs. The authors deduce that spatial positioning of N-glycans on one side of a conserved domain of hepsin, known as the scavenger receptor-rich cysteine domain, regulates calnexin selection for hepsin maturation and transport through the secretory pathway. If N-glycosylation is elsewhere on hepsin, then it is misfolded and has a prolonged accumulation with calnexin and BiP. This association coincides with the engagement of stress response pathways that sense glycoprotein misfolding. The topological considerations of N-glycosylation dissected by Sun et al. may help unravel how key sites of N-glycosylation sites required for protein folding and transport have evolved to select the lectin chaperone calnexin pathway for folding and quality control.

Keywords:  BiP; ER; N-glycosylation; UGGT; calnexin; hepsin; quality control; secretion

DOI:  https://doi.org/10.1111/febs.16779
Mol Ther. 2023 Apr 03. pii: S1525-0016(23)00161-2. [Epub ahead of print]

The unfolded protein response transcription factor XBP1s ameliorates Alzheimer's disease by improving synaptic function and proteostasis.

Claudia Duran-Aniotz, Natalia Poblete, Catalina Rivera-Krstulovic, Álvaro O Ardiles, Mei Li Díaz, Giovanni Tamburini, Carleen Mae P Sabusap, Yannis Gerakis, Felipe Cabral Miranda, Javier Diaz, Matias Fuentealba, Diego Arriagada, Ernesto Muñoz, Sandra Espinoza, Gabriela Martinez, Gabriel Quiroz, Danilo B Medinas, Darwin Contreras, Ricardo Piña, Mychael V Lourenco, Felipe C Ribeiro, Sergio T Ferreira, Carlos Rozas, Bernardo Morales, Lars Plate, Christian Gonzalez-Billault, Adrian G Palacios, Claudio Hetz.

Alteration in the buffering capacity of the proteostasis network is an emerging feature of Alzheimer's disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is the main adaptive pathway to cope with protein folding stress at the ER. Inositol requiring enzyme-1 (IRE1) operates as a central ER stress sensor, enabling the establishment of adaptive and repair programs through the control of the expression of the transcription factor X-Box binding protein 1 (XBP1). To artificially enforce the adaptive capacity of the UPR in the AD brain, we developed strategies to express the active form of XBP1 in the brain. Overexpression of XBP1 in the nervous system using transgenic mice reduced the load of amyloid deposits and preserved synaptic and cognitive function. Moreover, local delivery of XBP1 into the hippocampus of an AD mice using adeno-associated vectors improved different AD features. XBP1 expression corrected a large proportion of the proteomic alterations observed in the 5xFAD model, restoring the levels of several synaptic proteins and factors involved in actin cytoskeleton regulation and axonal growth. Our results illustrate the therapeutic potential of targeting UPR-dependent gene expression programs as a strategy to ameliorate AD features and sustain synaptic function.

DOI: https://doi.org/10.1016/j.ymthe.2023.03.028
BMC Biol. 2023 Apr 07. 21(1): 73

The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia.

Sadek Amhaz, Batiste Boëda, Mouna Chouchène, Sabrina Colasse, Florent Dingli, Damarys Loew, Julien Henri, Céline Prunier, Laurence Levy.

   BACKGROUND: E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-β signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain.
RESULTS: We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-β signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding.
CONCLUSIONS: Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.

Keywords:  E3 ubiquitin ligase; RING; RNF111; RNF165; SKIL; TGF-β; TOPORS; Thioredoxin; UBXN7

DOI:  https://doi.org/10.1186/s12915-023-01576-4
Front Mol Biosci. 2023 ;10 1155521

Reversible protein assemblies in the proteostasis network in health and disease.

Verena Kohler, Claes Andréasson.

  While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular dysfunction, stress and disease. During recent years, it has become clear that large aggregate-like protein condensates formed via liquid-liquid phase separation age into more solid aggregate-like particles that harbor misfolded proteins and are decorated by protein quality control factors. The constituent proteins of the condensates/aggregates are disentangled by protein disaggregation systems mainly based on Hsp70 and AAA ATPase Hsp100 chaperones prior to their handover to refolding and degradation systems. Here, we discuss the functional roles that condensate formation/aggregation and disaggregation play in protein quality control to maintain proteostasis and why it matters for understanding health and disease.

Keywords:  Hsp100; Hsp70; aggregate; biomolecular condensate; degradation; disaggregation; phase separation; refolding

DOI:  https://doi.org/10.3389/fmolb.2023.1155521
EMBO Rep. 2023 Apr 06. e57231

The fifth element: a novel eIF2α kinase in microglia.

Alessia Ruggieri.

The cellular integrated stress response (ISR) is a central signaling pathway that tunes translation initiation in response to a wide range of cellular insults to promote cell survival. The critical node of this regulation involves the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) by stress kinases. In this issue of EMBO reports, Wu et al (2023) report FAM69C as a novel eIF2α kinase that promotes ISR activation and stress granule (SG) assembly in microglia in response to oxidative stress. This work proposes a protective role for FAM69C and SGs in limiting damaging inflammatory responses commonly associated with neurodegenerative diseases.

DOI: https://doi.org/10.15252/embr.202357231
ACS Synth Biol. 2023 Apr 03.

Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox.

Dirk H Siepe, Lora K Picton, K Christopher Garcia.

  In recent years, targeted protein degradation (TPD) of plasma membrane proteins by hijacking the ubiquitin proteasome system (UPS) or the lysosomal pathway has emerged as a novel therapeutic avenue in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful in targeting cell surface receptors, these approaches are limited by the availability of suitable binders to generate heterobifunctional molecules. Here, we present the development of a nanobody (VHH)-based degradation toolbox termed REULR (Receptor Elimination by E3 Ubiquitin Ligase Recruitment). We generated human and mouse cross-reactive nanobodies against five transmembrane PA-TM-RING-type E3 ubiquitin ligases (RNF128, RNF130, RNF167, RNF43, and ZNRF3), covering a broad range and selectivity of tissue expression, with which we characterized the expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interactions with a variety of disease-relevant target receptors (EGFR, EPOR, and PD-1) by induced proximity, resulting in effective membrane clearance of the target receptor at varying levels. In addition, we designed E3 ligase self-degrading molecules, "fratricide" REULRs (RNF128, RNF130, RENF167, RNF43, and ZNRF3), that allow downregulation of one or several E3 ligases from the cell surface and consequently modulate receptor signaling strength. REULR molecules represent a VHH-based modular and versatile "mix and match" targeting strategy for the facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

Keywords:  E3 ligase; REULR; fratricide; induced proximity; nanobody; receptor; targeted protein degradation

DOI:  https://doi.org/10.1021/acssynbio.2c00587
Cell. 2023 Mar 31. pii: S0092-8674(23)00213-1. [Epub ahead of print]

Systemwide disassembly and assembly of SCF ubiquitin ligase complexes.

Kheewoong Baek, Daniel C Scott, Lukas T Henneberg, Moeko T King, Matthias Mann, Brenda A Schulman.

  Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of ∼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.

Keywords:  CAND1; CRL; E3 ligase; NEDD8; SCF; cryo-EM; cullin-RING ligase; protein complex assembly; proteomics; ubiquitin

DOI:  https://doi.org/10.1016/j.cell.2023.02.035
Am J Pathol. 2023 Apr 05. pii: S0002-9440(23)00121-9. [Epub ahead of print]

The dynamic role of endoplasmic reticulum stress in chronic liver disease.

Kaitlyn G Jackson, Grayson W Way, Jing Zeng, Marissa K Lipp, Huiping Zhou.

Chronic liver disease (CLD) is a major worldwide public health threat, with an estimated prevalence of 1.5 billion individuals suffering from CLD in 2020. Chronic activation of endoplasmic reticulum (ER) stress-related pathways is recognized to substantially contribute to the pathological progression of CLD. The ER is an intracellular organelle that folds proteins into their correct 3-dimensional shapes. ER-associated enzymes and chaperone proteins highly regulate this process. Perturbations in protein folding lead to misfolded or unfolded protein accumulation in the ER lumen, resulting in ER stress and concomitant activation of the unfolded protein response (UPR). The adaptive UPR is a set of signal transduction pathways evolved in mammalian cells that attempts to reestablish ER protein homeostasis by reducing protein load and increasing ER-associated degradation. However, maladaptive UPR responses in CLD occur due to prolonged UPR activation, leading to concomitant inflammation and cell death. This review assesses the current understanding of the cellular and molecular mechanisms that regulate ER stress and the UPR in the progression of different liver diseases and the potential pharmacological and biological interventions which target the UPR.

DOI: https://doi.org/10.1016/j.ajpath.2023.03.009
Life Sci Alliance. 2023 Jun;pii: e202201760. [Epub ahead of print]6(6):

The ubiquitin-like modifier FAT10 is degraded by the 20S proteasome in vitro but not in cellulo.

Franziska Oliveri, Steffen Johannes Keller, Heike Goebel, Gerardo Omar Alvarez Salinas, Michael Basler.

Ubiquitin-independent protein degradation via the 20S proteasome without the 19S regulatory particle has gained increasing attention over the last years. The degradation of the ubiquitin-like modifier FAT10 by the 20S proteasome was investigated in this study. We found that FAT10 was rapidly degraded by purified 20S proteasomes in vitro, which was attributed to the weak folding of FAT10 and the N-terminally disordered tail. To confirm our results in cellulo, we established an inducible RNA interference system in which the AAA-ATPase Rpt2 of the 19S regulatory particle is knocked down to impair the function of the 26S proteasome. Using this system, degradation of FAT10 in cellulo was strongly dependent on functional 26S proteasome. Our data indicate that in vitro degradation studies with purified proteins do not necessarily reflect biological degradation mechanisms occurring in cells and, therefore, cautious data interpretation is required when 20S proteasome function is studied in vitro.

DOI: https://doi.org/10.26508/lsa.202201760
J Cell Biol. 2023 May 01. pii: e202208147. [Epub ahead of print]222(5):

PERK prevents rhodopsin degradation during retinitis pigmentosa by inhibiting IRE1-induced autophagy.

Ning Zhao, Ning Li, Tao Wang.

Chronic endoplasmic reticulum (ER) stress is the underlying cause of many degenerative diseases, including autosomal dominant retinitis pigmentosa (adRP). In adRP, mutant rhodopsins accumulate and cause ER stress. This destabilizes wild-type rhodopsin and triggers photoreceptor cell degeneration. To reveal the mechanisms by which these mutant rhodopsins exert their dominant-negative effects, we established an in vivo fluorescence reporter system to monitor mutant and wild-type rhodopsin in Drosophila. By performing a genome-wide genetic screen, we found that PERK signaling plays a key role in maintaining rhodopsin homeostasis by attenuating IRE1 activities. Degradation of wild-type rhodopsin is mediated by selective autophagy of ER, which is induced by uncontrolled IRE1/XBP1 signaling and insufficient proteasome activities. Moreover, upregulation of PERK signaling prevents autophagy and suppresses retinal degeneration in the adRP model. These findings establish a pathological role for autophagy in this neurodegenerative condition and indicate that promoting PERK activity could be used to treat ER stress-related neuropathies, including adRP.

DOI: https://doi.org/10.1083/jcb.202208147
Dev Cell. 2023 Mar 29. pii: S1534-5807(23)00099-0. [Epub ahead of print]

The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia.

Swapnil Rohidas Shinde, David U Mick, Erika Aoki, Rachel B Rodrigues, Steven P Gygi, Maxence V Nachury.

  Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as hedgehog morphogens. Lysine 63-linked ubiquitin (UbK63) chains mark GPCRs for regulated removal from cilia, but the molecular basis of UbK63 recognition inside cilia remains elusive. Here, we show that the BBSome-the trafficking complex in charge of retrieving GPCRs from cilia-engages the ancestral endosomal sorting factor target of Myb1-like 2 (TOM1L2) to recognize UbK63 chains within cilia of human and mouse cells. TOM1L2 directly binds to UbK63 chains and the BBSome, and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin, and the GPCRs SSTR3, Smoothened, and GPR161 inside cilia. Furthermore, the single-cell alga Chlamydomonas also requires its TOM1L2 ortholog in order to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.

Keywords:  BBSome; ESCRT; GPCR; cilia; ubiquitin

DOI:  https://doi.org/10.1016/j.devcel.2023.03.003
Cell Rep. 2023 Apr 03. pii: S2211-1247(23)00350-9. [Epub ahead of print]42(4): 112339

SENP3 and USP7 regulate Polycomb-rixosome interactions and silencing functions.

Haining Zhou, Wenzhi Feng, Juntao Yu, Tiasha A Shafiq, Joao A Paulo, Jiuchun Zhang, Zhenhua Luo, Steven P Gygi, Danesh Moazed.

  The rixosome and PRC1 silencing complexes are associated with deSUMOylating and deubiquitinating enzymes, SENP3 and USP7, respectively. How deSUMOylation and deubiquitylation contribute to rixosome- and Polycomb-mediated silencing is not fully understood. Here, we show that the enzymatic activities of SENP3 and USP7 are required for silencing of Polycomb target genes. SENP3 deSUMOylates several rixosome subunits, and this activity is required for association of the rixosome with PRC1. USP7 associates with canonical PRC1 (cPRC1) and deubiquitinates the chromodomain subunits CBX2 and CBX4, and inhibition of USP activity results in disassembly of cPRC1. Finally, both SENP3 and USP7 are required for Polycomb- and rixosome-dependent silencing at an ectopic reporter locus. These findings demonstrate that SUMOylation and ubiquitination regulate the assembly and activities of the rixosome and Polycomb complexes and raise the possibility that these modifications provide regulatory mechanisms that may be utilized during development or in response to environmental challenges.

Keywords:  CP: Molecular biology; SENP3; SUMO; USP7; deSUMOylation; deubiquitination; epigenetics; gene silencing; heterochromatin; polycomb; rixosome; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2023.112339
mBio. 2023 Apr 05. e0019623

Contributions of Ccr4 and Gcn2 to the Translational Response of C. neoformans to Host-Relevant Stressors and Integrated Stress Response Induction.

Corey M Knowles, David Goich, Amanda L M Bloom, Murat C Kalem, John C Panepinto.

  In response to the host environment, the human pathogen Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host stress. In this study, we investigate the two events which comprise translatome reprogramming: the removal of abundant, pro-growth mRNAs from the translating pool, and the regulated entry of stress-responsive mRNAs into the translating pool. Removal of pro-growth mRNAs from the translating pool is controlled primarily by two regulatory mechanisms, repression of translation initiation via Gcn2, and decay mediated by Ccr4. We determined that translatome reprogramming in response to oxidative stress requires both Gcn2 and Ccr4, whereas the response to temperature requires only Ccr4. Additionally, we assessed ribosome collision in response to host-relevant stress and found that collided ribosomes accumulated during temperature stress but not during oxidative stress. The phosphorylation of eIF2α that occurred as a result of translational stress led us to investigate the induction of the integrated stress response (ISR). We found that eIF2α phosphorylation varied in response to the type and magnitude of stress, yet all tested conditions induced translation of the ISR transcription factor Gcn4. However, Gcn4 translation did not necessarily result in canonical Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress. In conclusion, this study begins to reveal the translational regulation in response to host-relevant stressors in an environmental fungus which is capable of adapting to the environment inside the human host. IMPORTANCE Cryptococcus neoformans is a human pathogen capable of causing devastating infections. It must rapidly adapt to changing environments as it leaves its niche in the soil and enters the human lung. Previous work has demonstrated a need to reprogram gene expression at the level of translation to promote stress adaptation. In this work, we investigate the contributions and interplay of the major mechanisms that regulate entry of new mRNAs into the pool (translation initiation) and the clearance of unneeded mRNAs from the pool (mRNA decay). One result of this reprogramming is the induction of the integrated stress response (ISR) regulon. Surprisingly, all stresses tested led to the production of the ISR transcription factor Gcn4, but not necessarily to transcription of ISR target genes. Furthermore, stresses result in differential levels of ribosome collisions, but these are not necessarily predictive of initiation repression as has been suggested in the model yeast.

Keywords:  integrated stress response; ribosome collision; stress adaptation; translational control

DOI:  https://doi.org/10.1128/mbio.00196-23
Front Cell Dev Biol. 2023 ;11 1089728

Normal tissue homeostasis and impairment of selective inflammatory responses in dendritic cells deficient for ATF6α.

Francisca Gutiérrez-Ballesteros, Jonathan Morales-Reyes, Dominique Fernández, Antonia Geisse, Amada Arcaya, Felipe Flores-Santibañez, María Rosa Bono, Fabiola Osorio.

  The initiation of adaptive immunity relies on the performance of dendritic cells (DCs), which are specialized leukocytes with professional antigen presenting capabilities. As such, the molecular mechanisms safeguarding DC homeostasis are matter of intense research. Sensors of the unfolded protein response (UPR) of the endoplasmic reticulum, a three-pronged signaling pathway that maintains the fidelity of the cellular proteome, have emerged as regulators of DC biology. The archetypical example is the IRE1/XBP1s axis, which supports DC development and survival of the conventional type 1 DC (cDC1) subtype. However, the role of additional UPR sensors in DC biology, such as the ATF6α branch, has not been clearly elucidated. Even though Xbp1 is transcriptionally induced by ATF6α under ER stress, it is unclear if cDCs also co-opt the ATF6α branch in tissues. Here, we examine the role of ATF6α in cDC homeostasis in vivo and upon innate stimulation in vitro. In steady state, animals lacking ATF6α in CD11c+ cells (Itgax Cre x Atf6 fl/fl mice) display normal cDC frequencies in spleen, intestine, liver, and lung. Also, ATF6α deficient cDCs express normal levels of Xbp1 mRNA and additional UPR components. However, a reduction of lung monocytes is observed in Itgax Cre x Atf6 fl/fl conditional deficient animals suggesting that ATF6α may play a role in the biology of monocyte subsets. Notably, in settings of DC activation, ATF6α contributes to the production of IL-12 and IL-6 to inflammatory stimuli. Thus, although ATF6α may be dispensable for tissue cDC homeostasis in steady state, the transcription factor plays a role in the acquisition of selective immunogenic features by activated DCs.

Keywords:  ATF6; IL-12; IL-6; dendritic cells; immunity; proinflammatory cytokines; tissues; unfolded protein response

DOI:  https://doi.org/10.3389/fcell.2023.1089728
Autophagy. 2023 Apr 04.

LC3-independent autophagy is vital to prevent TNF cytotoxicity.

Dario Priem, Jon Huyghe, Mathieu Jm Bertrand.

  The (macro)autophagy field is facing a paradigm shift after the recent discovery that cytosolic cargoes can still be selectively targeted to phagophores (the precursors to autophagosomes) even in the absence of LC3 or other Atg8-protein family members. Several in vitro studies have indeed reported on the existence of an unconventional selective autophagic pathway that involves the in-situ formation of an autophagosome around the cargo through the direct selective autophagy receptor-mediated recruitment of RB1CC1/FIP200, thereby bypassing the requirement of LC3. In an article recently published in Science, we demonstrate the physiological importance of this unconventional autophagic pathway in the context of TNF (tumor necrosis factor) signaling. We show that it promotes the degradation of the cytotoxic TNFRSF1A/TNFR1 (TNF receptor superfamily member 1A) complex II that assembles upon TNF sensing and thereby protects mice from TNFRSF1A-driven embryonic lethality and skin inflammation.

Keywords:  ATG9A; Apoptosis; LC3-independent autophagy; RB1CC1; TAX1BP1; cell death; embryogenesis; mouse development; skin disease; unconventional autophagy

DOI:  https://doi.org/10.1080/15548627.2023.2197760
J Biol Chem. 2023 Apr 01. pii: S0021-9258(23)00310-1. [Epub ahead of print] 104668

Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis.

Zhuangzhuang Zhao, Ling-Dong Xu, Fei Zhang, Qi-Zhang Liang, Yajuan Jiao, Fang-Shu Shi, Biao He, Pinglong Xu, Yao-Wei Huang.

  Inhibition of heat shock protein 90 (Hsp90), a prominent molecular chaperone, effectively limits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but little is known about any interaction between Hsp90 and SARS-CoV-2 proteins. Here, we systematically analyzed the effects of the chaperone isoforms Hsp90α and Hsp90β on individual SARS-CoV-2 viral proteins. Five SARS-CoV-2 proteins, namely nucleocapsid (N), membrane (M), and accessory proteins Orf3, Orf7a, and Orf7b were found to be novel clients of Hsp90β in particular. Pharmacological inhibition of Hsp90 with 17-DMAG results in N protein proteasome-dependent degradation. Hsp90 depletion-induced N protein degradation is independent of CHIP, a ubiquitin E3 ligase previously identified for Hsp90 client proteins, but alleviated by FBXO10, an E3 ligase identified by subsequent siRNA screening. We also provide evidence that Hsp90 depletion may suppress SARS-CoV-2 assembly partially through induced M or N degradation. Additionally, we found that GSDMD-mediated pyroptotic cell death triggered by SARS-CoV-2 was mitigated by inhibition of Hsp90. These findings collectively highlight a beneficial role for targeting of Hsp90 during SARS-CoV-2 infection, directly inhibiting virion production and reducing inflammatory injury by preventing the pyroptosis that contributes to severe SARS-CoV-2 disease.

Keywords:  Degradation; E3 ligase; Gasdermin D; Hsp90; Nucleoprotein; Pyroptosis; SARS-CoV-2

DOI:  https://doi.org/10.1016/j.jbc.2023.104668
J Cell Biol. 2023 May 01. pii: e201910133. [Epub ahead of print]222(5):

ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience.

Natalia Jiménez-Moreno, Madhu Kollareddy, Petros Stathakos, Joanna J Moss, Zuriñe Antón, Deborah K Shoemark, Richard B Sessions, Ralph Witzgall, Maeve Caldwell, Jon D Lane.

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.

DOI: https://doi.org/10.1083/jcb.201910133
Expert Opin Drug Discov. 2023 Apr;18(4): 363-370

PROTAC antibiotics: the time is now.

Jickky Palmae Sarathy, Courtney C Aldrich, Mei-Lin Go, Thomas Dick.

   INTRODUCTION: Novel antibiotics are needed to keep antibiotic resistance at bay and to improve treatment of the many drug-susceptible infections for which current therapies achieve poor cure rates. While revolutionizing human therapeutics, the concept of targeted protein degradation (TPD) by bifunctional proteolysis targeting chimeras (PROTACs) has not yet been applied to the discovery of antibiotics. A major obstacle precluding successful translation of this strategy to antibiotic development is that bacteria lack the E3 ligase-proteasome system exploited by human PROTACs to facilitate target degradation.
AREAS COVERED: The authors describe the serendipitous discovery of the first monofunctional target-degrading antibiotic pyrazinamide, supporting TPD as a viable and novel approach in antibiotic discovery. They then discuss the rational design, mechanism, and activity of the first bifunctional antibacterial target degrader BacPROTAC, enabling a generalizable approach to TPD in bacteria.
EXPERT OPINION: BacPROTACs demonstrate that linking a target directly to a bacterial protease complex can promote target degradation. BacPROTACs successfully bypass the 'middleman' E3 ligase, providing an entry strategy for the generation of antibacterial PROTACs. We speculate that antibacterial PROTACs will not only expand the target space but may also improve treatment by allowing dosage reduction, stronger bactericidal activity and activity against drug-tolerant 'persisters.'

Keywords:  Antibacterial drug discovery; proteolysis targeting chimeras; targeted protein degradation

DOI:  https://doi.org/10.1080/17460441.2023.2178413
Chembiochem. 2023 Apr 05. e202300163

Biophysical and computational approaches to study ternary complexes: A 'cooperative relationship' to rationalize targeted protein degradation.

Jake A Ward, Carles Perez-Lopez, Cristina Mayor-Ruiz.

  Degraders have illustrated that compound-induced proximity to E3 ubiquitin ligases can prompt the ubiquitination and degradation of disease-relevant proteins. Hence, this pharmacology is becoming a promising alternative and complement to available therapeutic interventions (e.g., inhibitors). Degraders rely on protein binding instead of inhibition and, hence, they hold the promise to broaden the druggable proteome. Biophysical and structural biology approaches have been the cornerstone of understanding and rationalizing degrader-induced ternary complex formation. Computational models have now started to harness the experimental data from these approaches with the aim to identify and rationally help design new degraders. This review outlines the current experimental and computational strategies used to study ternary complex formation and degradation and highlights the importance of effective crosstalk between these approaches in the advancement of the targeted protein degradation (TPD) field. As our understanding of the molecular features that govern drug-induced interactions grows, faster optimizations and superior therapeutic innovations for TPD and other proximity-inducing modalities are sure to follow.

Keywords:  Degraders; PROTACs; biophysical assays; computational strategies; molecular glues

DOI:  https://doi.org/10.1002/cbic.202300163
Nature. 2023 Apr 05.

The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Sumit Sen Santara, Dian-Jang Lee, Ângela Crespo, Jun Jacob Hu, Caitlin Walker, Xiyu Ma, Ying Zhang, Sourav Chowdhury, Karla F Meza-Sosa, Mercedes Lewandrowski, Haiwei Zhang, Marjorie Rowe, Arthur McClelland, Hao Wu, Caroline Junqueira, Judy Lieberman.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.

DOI: https://doi.org/10.1038/s41586-023-05912-0
Biochem J. 2023 Apr 05. pii: BCJ20220598. [Epub ahead of print]

ERK1/2 inhibitors act as monovalent degraders inducing ubiquitylation and proteasome-dependent turnover of ERK2, but not ERK1.

Kathryn Balmanno, Andrew M Kidger, Dominic P Byrne, Matthew J Sale, Nejma Nassman, Patrick A Eyers, Simon J Cook.

  Innate or acquired resistance to small molecule BRAF or MEK1/2 inhibitors (BRAFi or MEKi) typically arises through mechanisms that sustain or reinstate ERK1/2 activation. This has led to the development of a range of ERK1/2 inhibitors (ERKi) that either inhibit kinase catalytic activity (catERKi) or additionally prevent the activating pT-E-pY dual phosphorylation of ERK1/2 by MEK1/2 (dual-mechanism or dmERKi). Here we show that eight different ERKi (both catERKi or dmERKi) drive the turnover of ERK2, the most abundant ERK isoform, with little or no effect on ERK1. Thermal stability assays show that ERKi do not destabilise ERK2 (or ERK1) in vitro, suggesting that ERK2 turnover is a cellular consequence of ERKi binding. ERK2 turnover is not observed upon treatment with MEKi alone, suggesting it is ERKi binding to ERK2 that drives ERK2 turnover. However, MEKi pre-treatment, which blocks ERK2 pT-E-pY phosphorylation and dissociation from MEK1/2, prevents ERK2 turnover. ERKi treatment of cells drives the poly-ubiquitylation and proteasome-dependent turnover of ERK2 and pharmacological or genetic inhibition of Cullin-RING E3 ligases prevents this. Our results suggest that ERKi, including current clinical candidates, act as 'kinase degraders', driving the proteasome-dependent turnover of their major target, ERK2. This may be relevant to the suggestion of kinase-independent effects of ERK1/2 and the therapeutic use of ERKi.

Keywords:  ERK inhibitors; MEK; RAF; RAS; extracellular signal-regulated kinases; ubiquitin proteasome system

DOI:  https://doi.org/10.1042/BCJ20220598
ACS Chem Biol. 2023 Apr 05.

HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development.

Tiffany Tsang, Fidel Huerta, Yingpeng Liu, Jianwei Che, Rebecca J Metivier, Silas Ferrao, Katherine A Donovan, Lyn H Jones, Breanna L Zerfas, Radosław P Nowak.

The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cell-based quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.

DOI: https://doi.org/10.1021/acschembio.3c00084
FASEB J. 2023 05;37(5): e22902

Emerging evidence for poxvirus-mediated unfolded protein response: Lumpy skin disease virus maintains self-replication by activating PERK and IRE1 signaling.

Jinlong Tan, Yinju Liu, Fan Yang, Guohua Chen, Yongxiang Fang, Xiaobing He, Zhongzi Lou, Huaijie Jia, Zhizhong Jing, Weike Li.

  The monkeypox epidemic has attracted global attention to poxviruses. The cytoplasmic replication of poxviruses requires extensive protein synthesis, challenging the capacity of the endoplasmic reticulum (ER). However, the role of the ER in the life cycle of poxviruses is unclear. In this study, we demonstrate that infection with the lumpy skin disease virus (LSDV), a member of the poxvirus family, causes ER stress in vivo and in vitro, further facilitating the activation of the unfolded protein response (UPR). Although UPR activation aids in the restoration of the cellular environment, its significance in the LSDV life cycle remains unclear. Furthermore, the significance of ER imbalance for viral replication is also unknown. We show that LSDV replication is hampered by an unbalanced ER environment. In addition, we verify that the LSDV replication depends on the activation of PERK-eIF2α and IRE1-XBP1 signaling cascades rather than ATF6, implying that global translation and reduced XBP1 cleavage are deleterious to LSDV replication. Taken together, these findings indicate that LSDV is involved in the repression of global translational signaling, ER chaperone transcription, and ATF6 cleavage from the Golgi into the nucleus, thereby maintaining cell homeostasis; moreover, PERK and IRE1 activation contribute to LSDV replication. Our findings suggest that targeting UPR elements may be applied in response to infection from LSDV or even other poxviruses, such as monkeypox.

Keywords:  cell signaling; endoplasmic reticulum stress; lumpy skin disease virus; poxvirus; unfolded protein response

DOI:  https://doi.org/10.1096/fj.202300028R
Nat Cell Biol. 2023 Apr 06.

A lysosome membrane regeneration pathway depends on TBC1D15 and autophagic lysosomal reformation proteins.

Anshu Bhattacharya, Rukmini Mukherjee, Santosh Kumar Kuncha, Melinda Elaine Brunstein, Rajeshwari Rathore, Stephan Junek, Christian Münch, Ivan Dikic.

Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in cells depleted in TFEB and TFE3. We combined proximity-labelling-based proteomics, biochemistry and high-resolution microscopy to unravel a lysosomal membrane regeneration pathway that depends on ATG8, the lysosomal membrane protein LIMP2, the RAB7 GTPase-activating protein TBC1D15 and proteins required for autophagic lysosomal reformation, including dynamin-2, kinesin-5B and clathrin. Following lysosomal damage, LIMP2 acts as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 interacts with ATG8 proteins on damaged lysosomes and provides a scaffold to assemble and stabilize the autophagic lysosomal reformation machinery. This potentiates the formation of lysosomal tubules and subsequent dynamin-2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.

DOI: https://doi.org/10.1038/s41556-023-01125-9
Cell Rep. 2023 Apr 05. pii: S2211-1247(23)00356-X. [Epub ahead of print]42(4): 112345

Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control.

Yan Gao, Yousuf A Khan, Weike Mo, K Ian White, Matthew Perkins, Richard A Pfuetzner, Josef G Trapani, Axel T Brunger, Teresa Nicolson.

  The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. In vitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

Keywords:  CP: Neuroscience; NSF; SNARE complex; balance; deafness; hair cell; hearing; ribbon synapse; sensory deficit; zebrafish

DOI:  https://doi.org/10.1016/j.celrep.2023.112345
Proc Natl Acad Sci U S A. 2023 Apr 11. 120(15): e2207898120

eIF2Bδ blocks the integrated stress response and maintains eIF2B activity and cancer metastasis by overexpression in breast cancer stem cells.

Malavika Gupta, Beth A Walters, Olga Katsara, Karol Granados Blanco, Phillip A Geter, Robert J Schneider.

  Breast cancer (BC) metastasis involves cancer stem cells (CSCs) and their regulation by micro-RNAs (miRs), but miR targeting of the translation machinery in CSCs is poorly explored. We therefore screened miR expression levels in a range of BC cell lines, comparing non-CSCs to CSCs, and focused on miRs that target translation and protein synthesis factors. We describe a unique translation regulatory axis enacted by reduced expression of miR-183 in breast CSCs, which we show targets the eIF2Bδ subunit of guanine nucleotide exchange factor eIF2B, a regulator of protein synthesis and the integrated stress response (ISR) pathway. We report that reduced expression of miR-183 greatly increases eIF2Bδ protein levels, preventing strong induction of the ISR and eIF2α phosphorylation, by preferential interaction with P-eIF2α. eIF2Bδ overexpression is essential for BC cell invasion, metastasis, maintenance of metastases, and breast CSC expansion in animal models. Increased expression of eIF2Bδ, a site of action of the drug ISRIB that also prevents ISR signaling, is essential for breast CSC maintenance and metastatic capacity.

Keywords:  breast cancer; integrated stress response; metastasis; translational regulation; unfolded protein response

DOI:  https://doi.org/10.1073/pnas.2207898120
Nature. 2023 Apr 05.

De novo design of modular peptide-binding proteins by superhelical matching.

Kejia Wu, Hua Bai, Ya-Ting Chang, Rachel Redler, Kerrie E McNally, William Sheffler, T J Brunette, Derrick R Hicks, Tomos E Morgan, Tim J Stevens, Adam Broerman, Inna Goreshnik, Michelle DeWitt, Cameron M Chow, Yihang Shen, Lance Stewart, Emmanuel Derivery, Daniel Adriano Silva, Gira Bhabha, Damian C Ekiert, David Baker.

General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1-3. Here, inspired by natural and re-engineered protein-peptide systems4-11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.

DOI: https://doi.org/10.1038/s41586-023-05909-9
Nat Commun. 2023 Apr 05. 14(1): 1881

Oncogenic CALR mutant C-terminus mediates dual binding to the thrombopoietin receptor triggering complex dimerization and activation.

Nicolas Papadopoulos, Audrey Nédélec, Allison Derenne, Teodor Asvadur Şulea, Christian Pecquet, Ilyas Chachoua, Gaëlle Vertenoeil, Thomas Tilmant, Andrei-Jose Petrescu, Gabriel Mazzucchelli, Bogdan I Iorga, Didier Vertommen, Stefan N Constantinescu.

Calreticulin (CALR) frameshift mutations represent the second cause of myeloproliferative neoplasms (MPN). In healthy cells, CALR transiently and non-specifically interacts with immature N-glycosylated proteins through its N-terminal domain. Conversely, CALR frameshift mutants turn into rogue cytokines by stably and specifically interacting with the Thrombopoietin Receptor (TpoR), inducing its constitutive activation. Here, we identify the basis of the acquired specificity of CALR mutants for TpoR and define the mechanisms by which complex formation triggers TpoR dimerization and activation. Our work reveals that CALR mutant C-terminus unmasks CALR N-terminal domain, rendering it more accessible to bind immature N-glycans on TpoR. We further find that the basic mutant C-terminus is partially α-helical and define how its α-helical segment concomitantly binds acidic patches of TpoR extracellular domain and induces dimerization of both CALR mutant and TpoR. Finally, we propose a model of the tetrameric TpoR-CALR mutant complex and identify potentially targetable sites.

DOI: https://doi.org/10.1038/s41467-023-37277-3
Nat Commun. 2023 Apr 06. 14(1): 1941

Targeting USP2 regulation of VPRBP-mediated degradation of p53 and PD-L1 for cancer therapy.

Jingjie Yi, Omid Tavana, Huan Li, Donglai Wang, Richard J Baer, Wei Gu.

Since Mdm2 (Mouse double minute 2) inhibitors show serious toxicity in clinic studies, different approaches to achieve therapeutic reactivation of p53-mediated tumor suppression in cancers need to be explored. Here, we identify the USP2 (ubiquitin specific peptidase 2)-VPRBP (viral protein R binding protein) axis as an important pathway for p53 regulation. Like Mdm2, VPRBP is a potent repressor of p53 but VPRBP stability is controlled by USP2. Interestingly, the USP2-VPRBP axis also regulates PD-L1 (programmed death-ligand 1) expression. Strikingly, the combination of a small-molecule USP2 inhibitor and anti-PD1 monoclonal antibody leads to complete regression of the tumors expressing wild-type p53. In contrast to Mdm2, knockout of Usp2 in mice has no obvious effect in normal tissues. Moreover, no obvious toxicity is observed upon the USP2 inhibitor treatment in vivo as Mdm2-mediated regulation of p53 remains intact. Our study reveals a promising strategy for p53-based therapy by circumventing the toxicity issue.

DOI: https://doi.org/10.1038/s41467-023-37617-3
Autophagy. 2023 Apr 04.

Hepatocyte CD36 modulates UBQLN1-mediated proteasomal degradation of autophagic SNARE proteins contributing to septic liver injury.

Yanping Li, Jingyuan Xu, Weiting Chen, Xingxing Wang, Zhibo Zhao, Yuqi Li, Linkun Zhang, Junkui Jiao, Qin Yang, Qiuying Ding, Ping Yang, Li Wei, Yao Chen, Yaxi Chen, Xiong Z Ruan, Lei Zhao.

  Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury.

Keywords:  CD36; autophagy; liver injury; proteasomal degradation; ubiquilin 1

DOI:  https://doi.org/10.1080/15548627.2023.2196876