bims-proteo 2024-10-20 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024‒10‒20
34 papers selected by
Eric Chevet, INSERM

Principles of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2.
Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.
Interactors and neighbors of ULK1 complex members.
AN ATYPICAL E3 LIGASE SAFEGUARDS THE RIBOSOME DURING NUTRIENT STRESS.
O-GlcNAcylation of ribosome-associated proteins is concomitant with translational reprogramming during proteotoxic stress.
The neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis in Caenorhabditis elegans.
Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders.
A machine learning approach uncovers principles and determinants of eukaryotic ribosome pausing.
Allosteric degraders induce CRL5 ASB8 mediated degradation of XPO1.
Early steps of protein disaggregation by Hsp70 chaperone and class B J-domain proteins are shaped by Hsp110.
Peroxisomal import stress activates integrated stress response and inhibits ribosome biogenesis.
TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport.
Redirecting the pioneering function of FOXA1 with covalent small molecules.
USP7 protects TFEB from proteasome-mediated degradation.
Autophagy adaptors mediate Parkin-dependent mitophagy by forming sheet-like liquid condensates.
The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature.
Mitophagy as a guardian against cellular aging.
Nuclear p62 condensates stabilize the promyelocytic leukemia nuclear bodies by sequestering their ubiquitin ligase RNF4.
Ubiquitin ligase MARCH5 controls the formation of mitochondria-derived pre-peroxisomes.
Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates.
Reticulophagy and viral infection.
Targeting IRE1α reprograms the tumor microenvironment and enhances anti-tumor immunity in prostate cancer.
Defective ribosome assembly impairs leukemia progression in a murine model of acute myeloid leukemia.
Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins.
Global profiling of protein complex dynamics with an experimental library of protein interaction markers.
The genetic architecture of protein interaction affinity and specificity.
IRE1α silences dsRNA to prevent taxane-induced pyroptosis in triple-negative breast cancer.
Toward Understanding the Mechanism of Client-Selective Small Molecule Inhibitors of the Sec61 Translocon.
ELiAH: the atlas of E3 ligases in human tissues for targeted protein degradation with reduced off-target effect.
Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma.
The translatome of glioblastoma.
Fluorescence Lifetime-Assisted Probing of Protein Aggregation with sub-Organellar Resolution.
The ribosome profiling landscape of yeast reveals a high diversity in pervasive translation.
Spatial proteomics identifies JAKi as treatment for a lethal skin disease.

Nat Commun. 2024 Oct 12. 15(1): 8829

Principles of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2.

Daniel C Scott, Suresh Dharuman, Elizabeth Griffith, Sergio C Chai, Jarrid Ronnebaum, Moeko T King, Rajendra Tangallapally, Chan Lee, Clifford T Gee, Lei Yang, Yong Li, Victoria C Loudon, Ha Won Lee, Jason Ochoada, Darcie J Miller, Thilina Jayasinghe, Joao A Paulo, Stephen J Elledge, J Wade Harper, Taosheng Chen, Richard E Lee, Brenda A Schulman.

PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2's U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3BD2. Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases.

DOI: https://doi.org/10.1038/s41467-024-52966-3
J Cell Biol. 2025 Jan 06. pii: e202312141. [Epub ahead of print]224(1):

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

Pei Yee Tey, Almut Dufner, Klaus-Peter Knobeloch, Jonathan N Pruneda, Michael J Clague, Sylvie Urbé.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

DOI: https://doi.org/10.1083/jcb.202312141
Autophagy. 2024 Oct 14. 1-3

Interactors and neighbors of ULK1 complex members.

Devanarayanan Siva Sankar, Joern Dengjel.

  The ULK1 kinase complex plays a crucial role in autophagosome biogenesis. To identify interactors or regulators of ULK1 complex assembly influencing autophagosome biogenesis, we performed an interaction proteomics screen. Employing both affinity purification and proximity labeling of N- and C-terminal tagged fusion proteins coupled to quantitative mass spectrometry, we identified 317 high-confidence interactors or neighbors of the four ULK1 complex members, including both member-specific and common interactors. Interactions with selective macroautophagy/autophagy receptors indicate the activation of selective autophagy pathways by 90 min of nutrient starvation. Focusing on the ULK1 effector protein BAG2, a common interactor identified by both approaches, we highlight that ULK1 phosphorylates BAG2, supporting the localization of the scaffold and autophagy inducer AMBRA1 to the ER, thereby positively regulating autophagy initiation.Abbreviation: AMBRA1: autophagy and beclin 1 regulator 1; ATG: autophagy related; ER: endoplasmic reticulum; HA: hemagglutinin; KD: knockdown; KO: knockout; MS: mass spectrometry; PTM: posttranslational modification; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Keywords:  Affinity purification; kinase; mass spectrometry; miniturbo; proteomics; proximity labeling

DOI:  https://doi.org/10.1080/15548627.2024.2414386
bioRxiv. 2024 Oct 11. pii: 2024.10.10.617692. [Epub ahead of print]

AN ATYPICAL E3 LIGASE SAFEGUARDS THE RIBOSOME DURING NUTRIENT STRESS.

Todd Douglas, Jiasheng Zhang, Zhiping Wu, Kyrillos Abdallah, Meghan McReynolds, Wendy V Gilbert, Kazuhiro Iwai, Junmin Peng, Lawrence H Young, Craig M Crews.

Metabolic stress must be effectively mitigated for the survival of cells and organisms. Ribosomes have emerged as signaling hubs that sense metabolic perturbations and coordinate responses that either restore homeostasis or trigger cell death. As yet, the mechanisms governing these cell fate decisions are not well understood. Here, we report an unexpected role for the atypical E3 ligase HOIL-1 in safeguarding the ribosome. We find HOIL-1 mutations associated with cardiomyopathy broadly sensitize cells to nutrient and translational stress. These signals converge on the ribotoxic stress sentinel ZAKα. Mechanistically, mutant HOIL-1 excludes a ribosome quality control E3 ligase from its functional complex and remodels the ribosome ubiquitin landscape. This quality control failure renders glucose starvation ribotoxic, precipitating a ZAKα-ATF4-xCT-driven noncanonical cell death. We further show HOIL-1 loss exacerbates cardiac dysfunction under pressure overload. These data reveal an unrecognized ribosome signaling axis and a molecular circuit controlling cell fate during nutrient stress.

DOI: https://doi.org/10.1101/2024.10.10.617692
J Biol Chem. 2024 Oct 10. pii: S0021-9258(24)02379-2. [Epub ahead of print] 107877

O-GlcNAcylation of ribosome-associated proteins is concomitant with translational reprogramming during proteotoxic stress.

Quira Zeidan, Jie L Tian, Junfeng Ma, Farzad Eslami, Gerald W Hart.

  Protein O-GlcNAc modification, similar to phosphorylation, supports cell survival by regulating key processes like transcription, cell division, trafficking, signaling, and stress tolerance. However, its role in protein homeostasis, particularly in protein synthesis, folding, and degradation remains poorly understood. Our previous research shows that O-GlcNAc cycling enzymes associate with the translation machinery during protein synthesis and modify ribosomal proteins. Protein translation is closely linked to 26S proteasome activity, which recycles amino acids and clears misfolded proteins during stress, preventing aggregation and cell death. In this study, we demonstrate that pharmacological perturbation of the proteasome-like that used in cancer treatment- leads to the increased abundance of OGT and OGA in a ribosome-rich fraction, concurrent with O-GlcNAc modification of core translational and ribosome-associated proteins. This interaction is synchronous with eIF2α-dependent translational reprogramming. We also found that protein ubiquitination depends partly on O-GlcNAc metabolism in MEFs, as OGT-depleted cells show decreased ubiquitination under stress. Using an O-GlcNAc-peptide enrichment strategy followed by LC-MS/MS, we identified 84 unique O-GlcNAc sites across 55 proteins, including ribosomal proteins, nucleolar factors, and the 70-kDa heat shock protein family. Hsp70 and OGT colocalize with the translational machinery in an RNA-independent manner, aiding in partial protein translation recovery during sustained stress. O-GlcNAc cycling on ribosome-associated proteins collaborates with Hsp70 to restore protein synthesis during proteotoxicity, suggesting a role in tumor resistance to proteasome inhibitors.

Keywords:  O-GlcNAc; O-GlcNAc transferase; O-GlcNAcase; proteasome; proteotoxicity; ribosome; translation; ubiquitination

DOI:  https://doi.org/10.1016/j.jbc.2024.107877
Proc Natl Acad Sci U S A. 2024 Oct 22. 121(43): e2403906121

The neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis in Caenorhabditis elegans.

Shane K B Taylor, Jessica H Hartman, Bhagwati P Gupta.

  The conserved mesencephalic astrocyte-derived neurotrophic factor (MANF) is known for protecting dopaminergic neurons and functioning in various other tissues. Previously, we showed that Caenorhabditis elegans manf-1 null mutants exhibit defects such as increased endoplasmic reticulum (ER) stress, dopaminergic neurodegeneration, and abnormal protein aggregation. These findings suggest an essential role for MANF in cellular processes. However, the mechanisms by which intracellular and extracellular MANF regulate broader cellular functions remain unclear. We report a unique mechanism of action for MANF-1 that involves the transcription factor HLH-30/TFEB-mediated signaling to regulate autophagy and lysosomal function. Multiple transgenic strains overexpressing MANF-1 showed extended lifespan of animals, reduced protein aggregation, and improved neuronal survival. Using fluorescently tagged MANF-1, we observed tissue-specific localization of the protein, which was dependent on the ER retention signal. Further subcellular analysis showed that MANF-1 localizes within cells to the lysosomes and utilizes the endosomal pathway. Consistent with the lysosomal localization, our transcriptomic study of MANF-1 and analyses of autophagy regulators demonstrated that MANF-1 promotes proteostasis by regulating autophagic flux and lysosomal activity. Collectively, our findings establish MANF as a critical regulator of stress response, proteostasis, and aging.

Keywords:  ER stress; MANF-1; longevity; nematode; proteostasis

DOI:  https://doi.org/10.1073/pnas.2403906121
Nat Commun. 2024 Oct 15. 15(1): 8885

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders.

Alena Kroupova, Valentina A Spiteri, Zoe J Rutter, Hirotake Furihata, Darren Darren, Sarath Ramachandran, Sohini Chakraborti, Kevin Haubrich, Julie Pethe, Denzel Gonzales, Andre J Wijaya, Maria Rodriguez-Rios, Manon Sturbaut, Dylan M Lynch, William Farnaby, Mark A Nakasone, David Zollman, Alessio Ciulli.

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

DOI: https://doi.org/10.1038/s41467-024-52871-9
Sci Adv. 2024 Oct 18. 10(42): eado0738

A machine learning approach uncovers principles and determinants of eukaryotic ribosome pausing.

Mauricio Aguilar Rangel, Kevin Stein, Judith Frydman.

Nonuniform local translation speed dictates diverse protein biogenesis outcomes. To unify known and uncover unknown principles governing eukaryotic elongation rate, we developed a machine learning pipeline to analyze RiboSeq datasets. We find that the chemical nature of the incoming amino acid determines how codon optimality influences elongation rate, with hydrophobic residues more dependent on transfer RNA (tRNA) levels than charged residues. Unexpectedly, we find that wobble interactions exert a widespread effect on elongation pausing, with wobble-mediated decoding being slower than Watson-Crick decoding, irrespective of tRNA levels. Applying our ribosome pausing principles to ribosome collisions reveals that disomes arise upon apposition of fast-decoding and slow-decoding signatures. We conclude that codon choice and tRNA pools are evolutionarily constrained to harmonize elongation rate with cotranslational folding while minimizing wobble pairing and deleterious stalling.

DOI: https://doi.org/10.1126/sciadv.ado0738
bioRxiv. 2024 Oct 11. pii: 2024.10.07.617049. [Epub ahead of print]

Allosteric degraders induce CRL5 ASB8 mediated degradation of XPO1.

Casey Elizabeth Hudman-Wing, Ho Yee Joyce Fung, Tolga Cagatay, Bert Kwanten, Ashley B Niesman, Mehdi Gharghabi, Brecht Permentier, Binita Shakya, Sharon Shacham, Yosef Landesman, Rosa Lapalombella, Dirk Daelemans, Yuh Min Chook.

The nuclear export receptor Exportin 1 (XPO1/CRM1) is often overexpressed in cancer cells resulting in aberrant localization of many cancer-related protein cargoes. The XPO1 inhibitor and cancer drug selinexor (KPT-330), and its analog KPT-185, block XPO1-cargo binding thereby restoring cargo localization. Selinexor binding induces cullin-RING E3 ubiquitin ligase (CRL) substrate receptor ASB8-mediated XPO1 degradation. Here we reveal the mechanism of inhibitor-XPO1 engagement by CRL5ASB8. Cryogenic electron microscopy (cryo-EM) structures show ASB8 binding to a large surface of selinexor/KPT-185-XPO1 that includes a three-dimensional degron unique to the drug-bound exportin. The structure explains weak XPO1-ASB8 binding in the absence of selinexor/KPT-185 that is unproductive for proteasomal degradation, and the substantial affinity increase upon selinexor/KPT-185 conjugation, which results in CRL5 ASB8 -mediated XPO1 ubiquitination. In contrast to previously characterized small molecule degraders, which all act as molecular glues, selinexor/KPT-185 binds extensively to XPO1 but hardly contacts ASB8. Instead, selinexor/KPT-185 binds XPO1 and stabilizes a unique conformation of the NES/inhibitor-binding groove that binds ASB8. Selinexor/KPT-185 is an allosteric degrader. We have explained how drug-induced protein degradation is mediated by a CRL5 system through an allosteric rather than a molecular glue mechanism, expanding the modes of targeted protein degradation beyond the well-known molecular glues of CRL4.

DOI: https://doi.org/10.1101/2024.10.07.617049
Elife. 2024 Oct 15. pii: RP94795. [Epub ahead of print]13

Early steps of protein disaggregation by Hsp70 chaperone and class B J-domain proteins are shaped by Hsp110.

Wiktoria Sztangierska, Hubert Wyszkowski, Maria Pokornowska, Klaudia Kochanowicz, Michal Rychłowski, Krzysztof Liberek, Agnieszka Kłosowska.

  Hsp70 is a key cellular system counteracting protein misfolding and aggregation, associated with stress, ageing, and disease. Hsp70 solubilises aggregates and aids protein refolding through substrate binding and release cycles regulated by co-chaperones: J-domain proteins (JDPs) and nucleotide exchange factors (NEFs). Here, we elucidate the collaborative impact of Hsp110 NEFs and different JDP classes throughout Hsp70-dependent aggregate processing. We show that Hsp110 plays a major role at initial stages of disaggregation, determining its final efficacy. The NEF catalyses the recruitment of thick Hsp70 assemblies onto aggregate surface, which modifies aggregates into smaller species more readily processed by chaperones. Hsp70 stimulation by Hsp110 is much stronger with class B than class A JDPs and requires the auxiliary interaction between class B JDP and the Hsp70 EEVD motif. Furthermore, we demonstrate for the first time that Hsp110 disrupts the JDP-Hsp70 interaction. Such destabilisation of chaperone complexes at the aggregate surface might improve disaggregation, but also lead to the inhibition above the sub-stoichiometric Hsp110 optimum. Thus, balanced interplay between the co-chaperones and Hsp70 is critical to unlock its disaggregating potential.

Keywords:  S. cerevisiae; biochemistry; chemical biology; heat shock protein; homo sapiens; protein aggregation; protein folding; protein quality control; stress

DOI:  https://doi.org/10.7554/eLife.94795
PNAS Nexus. 2024 Oct;3(10): pgae429

Peroxisomal import stress activates integrated stress response and inhibits ribosome biogenesis.

Jinoh Kim, Kerui Huang, Pham Thuy Tien Vo, Ting Miao, Jacinta Correia, Ankur Kumar, Mirre J P Simons, Hua Bai.

  Impaired organelle-specific protein import triggers a variety of cellular stress responses, including adaptive pathways to balance protein homeostasis. Most of the previous studies focus on the cellular stress response triggered by misfolded proteins or defective protein import in the endoplasmic reticulum or mitochondria. However, little is known about the cellular stress response to impaired protein import in the peroxisome, an understudied organelle that has recently emerged as a key signaling hub for cellular and metabolic homeostasis. To uncover evolutionarily conserved cellular responses upon defective peroxisomal import, we carried out a comparative transcriptomic analysis on fruit flies with tissue-specific peroxin knockdown and human HEK293 cells expressing dominant-negative PEX5C11A. Our RNA-seq results reveal that defective peroxisomal import upregulates integrated stress response (ISR) and downregulates ribosome biogenesis in both flies and human cells. Functional analyses confirm that impaired peroxisomal import induces eIF2α phosphorylation and ATF4 expression. Loss of ATF4 exaggerates cellular damage upon peroxisomal import defects, suggesting that ATF4 activation serves as a cellular cytoprotective mechanism upon peroxisomal import stress. Intriguingly, we show that peroxisomal import stress decreases the expression of rRNA processing genes and inhibits early pre-rRNA processing, which leads to the accumulation of 47S precursor rRNA and reduction of downstream rRNA intermediates. Taken together, we identify ISR activation and ribosome biogenesis inhibition as conserved adaptive stress responses to defective peroxisomal import and uncover a novel link between peroxisomal dysfunction and rRNA processing.

Keywords:  PEX5; early rRNA processing; integrated stress response; peroxisomal import stress; ribosome biogenesis

DOI:  https://doi.org/10.1093/pnasnexus/pgae429
Nat Commun. 2024 Oct 19. 15(1): 9026

TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport.

Hongyi Wu, Loo Chien Wang, Belle M Sow, Damien Leow, Jin Zhu, Kathryn M Gallo, Kathleen Wilsbach, Roshni Gupta, Lyle W Ostrow, Crystal J J Yeo, Radoslaw M Sobota, Rong Li.

Protein aggregation plays key roles in age-related degenerative diseases, but how different proteins coalesce to form inclusions that vary in composition, morphology, molecular dynamics and confer physiological consequences is poorly understood. Here we employ a general reporter based on mutant Hsp104 to identify proteins forming aggregates in human cells under common proteotoxic stress. We identify over 300 proteins that form different inclusions containing subsets of aggregating proteins. In particular, TDP43, implicated in Amyotrophic Lateral Sclerosis (ALS), partitions dynamically between two distinct types of aggregates: stress granule and a previously unknown non-dynamic (solid-like) inclusion at the ER exit sites (ERES). TDP43-ERES co-aggregation is induced by diverse proteotoxic stresses and observed in the motor neurons of ALS patients. Such aggregation causes retention of secretory cargos at ERES and therefore delays ER-to-Golgi transport, providing a link between TDP43 aggregation and compromised cellular function in ALS patients.

DOI: https://doi.org/10.1038/s41467-024-52706-7
Mol Cell. 2024 Oct 09. pii: S1097-2765(24)00780-9. [Epub ahead of print]

Redirecting the pioneering function of FOXA1 with covalent small molecules.

Sang Joon Won, Yuxiang Zhang, Christopher J Reinhardt, Lauren M Hargis, Nicole S MacRae, Kristen E DeMeester, Evert Njomen, Jarrett R Remsberg, Bruno Melillo, Benjamin F Cravatt, Michael A Erb.

  Pioneer transcription factors (TFs) bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic compounds that stereoselectively and site-specifically bind the pioneer TF forkhead box protein A1 (FOXA1) at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the ligands relax the canonical DNA-binding preference of FOXA1 by strengthening interactions with suboptimal sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

Keywords:  ATAC-seq; ChIP-seq; FOXA1; activity-based protein profiling; chromatin; covalent; cysteine; pioneer transcription factor; proteomics

DOI:  https://doi.org/10.1016/j.molcel.2024.09.024
Cell Rep. 2024 Oct 15. pii: S2211-1247(24)01223-3. [Epub ahead of print]43(11): 114872

USP7 protects TFEB from proteasome-mediated degradation.

Swati Keshri, Mariella Vicinanza, Michael Takla, David C Rubinsztein.

  The transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy. We identify a distinct nuclear interactome of TFEB, with ubiquitin-specific protease 7 (USP7) emerging as a key post-translational modulator of TFEB. Genetic depletion and inhibition of USP7 reveal its critical role in preserving TFEB stability within both nuclear and cytoplasmic compartments. Specifically, USP7 is identified as the deubiquitinase responsible for removing the K48-linked polyubiquitination signal from TFEB at lysine residues K116, K264, and K274, thereby preventing its proteasomal degradation. Functional assays demonstrate the involvement of USP7 in preserving TFEB-mediated transcriptional responses to nutrient deprivation while also modulating autophagy flux and lysosome biogenesis. As USP7 is a deubiquitinase that protects TFEB from proteasomal degradation, these findings provide the foundation for therapeutic targeting of the USP7-TFEB axis in conditions characterized by TFEB dysregulation and metabolic abnormalities, particularly in certain cancers.

Keywords:  CP: Cell biology; CP: Molecular biology; TFEB; USP7; autophagy; lysosomal biogenesis; post-translational modifications; proteasomal degradation; ubiquitination

DOI:  https://doi.org/10.1016/j.celrep.2024.114872
EMBO J. 2024 Oct 17.

Autophagy adaptors mediate Parkin-dependent mitophagy by forming sheet-like liquid condensates.

Zi Yang, Saori R Yoshii, Yuji Sakai, Jing Zhang, Haruka Chino, Roland L Knorr, Noboru Mizushima.

  During PINK1- and Parkin-mediated mitophagy, autophagy adaptors are recruited to damaged mitochondria to promote their selective degradation. Autophagy adaptors such as optineurin (OPTN) and NDP52 facilitate mitophagy by recruiting the autophagy-initiation machinery, and assisting engulfment of damaged mitochondria through binding to ubiquitinated mitochondrial proteins and autophagosomal ATG8 family proteins. Here, we demonstrate that OPTN and NDP52 form sheet-like phase-separated condensates with liquid-like properties on the surface of ubiquitinated mitochondria. The dynamic and liquid-like nature of OPTN condensates is important for mitophagy activity, because reducing the fluidity of OPTN-ubiquitin condensates suppresses the recruitment of ATG9 vesicles and impairs mitophagy. Based on these results, we propose a dynamic liquid-like, rather than a stoichiometric, model of autophagy adaptors to explain the interactions between autophagic membranes (i.e., ATG9 vesicles and isolation membranes) and mitochondrial membranes during Parkin-mediated mitophagy. This model underscores the importance of liquid-liquid phase separation in facilitating membrane-membrane contacts, likely through the generation of capillary forces.

Keywords:  Autophagy; Liquid–Liquid Phase Separation; Mitophagy; Optineurin; Wetting

DOI:  https://doi.org/10.1038/s44318-024-00272-5
EMBO J. 2024 Oct 14.

The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature.

Yasuhisa Sakamoto, Akiyoshi Uezu, Koji Kikuchi, Jangmi Kang, Eiko Fujii, Toshiro Moroishi, Shiro Suetsugu, Hiroyuki Nakanishi.

  The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates membrane sorting during endocytosis through the ubiquitination of cargo molecules such as the epithelial sodium channel (ENaC). Nedd4L is catalytically autoinhibited by an intramolecular interaction between its C2 and HECT domains, but the protein's activation mechanism is poorly understood. Here, we show that Nedd4L activation is linked to membrane shape by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. FCHO2 was required for the Nedd4L-mediated ubiquitination and endocytosis of ENaC, with Nedd4L co-localizing with FCHO2 at clathrin-coated pits. In cells, Nedd4L was specifically recruited to, and activated by, the FCHO2 BAR domain. Furthermore, we reconstituted FCHO2-induced recruitment and activation of Nedd4L in vitro. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain, with this curvature directly activating Nedd4L by relieving its autoinhibition. Thus, we show for the first time a specific function (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.

Keywords:  Clathrin; Endocytosis; FCHO2; Membrane Curvature; Nedd4L

DOI:  https://doi.org/10.1038/s44318-024-00268-1
Autophagy. 2024 Oct 14. 1-3

Mitophagy as a guardian against cellular aging.

Tetsushi Kataura, Niall Wilson, Gailing Ma, Viktor I Korolchuk.

  Mitophagy, the selective autophagic clearance of damaged mitochondria, is considered vital for maintaining mitochondrial quality and cellular homeostasis; however, its molecular mechanisms, particularly under basal conditions, and its role in cellular physiology remain poorly characterized. We recently demonstrated that basal mitophagy is a key feature of primary human cells and is downregulated by immortalization, suggesting its dependence on the primary cell state. Mechanistically, we demonstrated that the PINK1-PRKN-SQSTM1 pathway regulates basal mitophagy, with SQSTM1 sensing superoxide-enriched mitochondria through its redox-sensitive cysteine residues, which mediate SQSTM1 oligomerization and mitophagy activation. We developed STOCK1N-57534, a small molecule that targets and promotes this SQSTM1 activation mechanism. Treatment with STOCK1N-57534 reactivates mitophagy downregulated in senescent and naturally aged donor-derived primary cells, improving cellular senescence(-like) phenotypes. Our findings highlight that basal mitophagy is protective against cellular senescence and aging, positioning its pharmacological reactivation as a promising anti-aging strategy.Abbreviation: IR: ionizing radiation; ROS: reactive oxygen species; SARs: selective autophagy receptors.

Keywords:  Aging; SQSTM1/p62; autophagy; mitochondria; mitophagy; senescence

DOI:  https://doi.org/10.1080/15548627.2024.2414461
Proc Natl Acad Sci U S A. 2024 Oct 22. 121(43): e2414377121

Nuclear p62 condensates stabilize the promyelocytic leukemia nuclear bodies by sequestering their ubiquitin ligase RNF4.

Afu Fu, Zhiwen Luo, Tamar Ziv, Xinyu Bi, Chen Lulu-Shimron, Victoria Cohen-Kaplan, Aaron Ciechanover.

  Liquid-liquid phase separation has emerged as a crucial mechanism driving the formation of membraneless biomolecular condensates, which play important roles in numerous cellular processes. These condensates, found both in the nucleus and cytoplasm, are formed through multivalent, low-affinity interactions between various molecules. P62-containing condensates serve, among other functions, as proteolytic hubs for the ubiquitin-proteasome system. In this study, we investigated the dynamic interplay between nuclear p62 condensates and promyelocytic nuclear bodies (PML-NBs). We show that p62 condensates stabilize PML-NBs under both basal conditions and following exposure to arsenic trioxide which stimulates their degradation. We further show that this effect on the stability of PML-NBs is due to sequestration of their ubiquitin E3 ligase RNF4 in the p62 condensates with subsequent rapid degradation of the ligase. The sequestration of the ligase is made possible by association between the proline-rich domain of the PML protein and the PB1 domain of p62, which results in the formation of a PML-NB shell around the p62 condensates. Importantly, these hybrid structures do not undergo fusion and mixing of their contents which leaves unsolved the mechanism of sequestration of RNF4 in the condensates. These findings suggest an additional possible mechanism of PML-NB as a tumor suppressor which is mediated via interactions between different biomolecular condensates.

Keywords:  PML-NB; RNF4; p62 condensates; protein degradation; ubiquitin

DOI:  https://doi.org/10.1073/pnas.2414377121
Dev Cell. 2024 Oct 14. pii: S1534-5807(24)00600-2. [Epub ahead of print]

Ubiquitin ligase MARCH5 controls the formation of mitochondria-derived pre-peroxisomes.

Jun Zheng, Jing Chen, Zhihe Cao, Kaichen Wu, Jinhui Wang, Yusong Guo, Min Zhuang.

  Peroxisome biogenesis involves two pathways: growth and division from pre-existing mature peroxisomes and de novo biogenesis from the endoplasmic reticulum, with a contribution from mitochondria, particularly in human peroxisome-deficient cells. However, the essential components that control peroxisome de novo biogenesis are largely unknown. Dual organelle localized ubiquitin ligase MARCH5 functions on peroxisomes to control pexophagy. Here, we show that mitochondria-localized MARCH5 is essential for the formation of vesicles in the de novo biogenesis of peroxisomes from mitochondria in human cell lines. Loss of MARCH5 specifically impedes the budding of PEX3-containing vesicles from mitochondria, thereby blocking the formation of pre-peroxisomes. Overall, our study highlights the function of MARCH5 for mitochondria-derived pre-peroxisomes, emphasizing MARCH5 as one regulator to maintain peroxisome homeostasis.

Keywords:  MARCH5; MDVs; PEX3; de nono biogenesis; mitochondria-derived vesicles; peroxisome

DOI:  https://doi.org/10.1016/j.devcel.2024.09.029
J Cell Biol. 2025 Jan 06. pii: e202311105. [Epub ahead of print]224(1):

Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates.

Leyla E Fahim, Joshua M Marcus, Noah D Powell, Zachary A Ralston, Katherine Walgamotte, Eleonora Perego, Giuseppe Vicidomini, Alessandro Rossetta, Jason E Lee.

Ribonucleoprotein (RNP) condensates partition RNA and protein into multiple liquid phases. The multiphasic feature of condensate-enriched components creates experimental challenges for distinguishing membraneless condensate functions from the surrounding dilute phase. We combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering and segmentation to resolve condensates from the dilute phase. Condensate-specific lifetimes were used to track protein-protein interactions by measuring FLIM-Förster resonance energy transfer (FRET). We used condensate FLIM-FRET to evaluate whether mRNA decapping complex subunits can form decapping-competent interactions within P-bodies. Condensate FLIM-FRET revealed the presence of core subunit interactions within P-bodies under basal conditions and the disruption of interactions between the decapping enzyme (Dcp2) and a critical cofactor (Dcp1A) during oxidative stress. Our results show a context-dependent plasticity of the P-body interaction network, which can be rewired within minutes in response to stimuli. Together, our FLIM-based approaches provide investigators with an automated and rigorous method to uncover and track essential protein-protein interaction dynamics within RNP condensates in live cells.

DOI: https://doi.org/10.1083/jcb.202311105
Autophagy. 2024 Oct 12.

Reticulophagy and viral infection.

Alexa Wilson, Craig McCormick.

  All viruses are obligate intracellular parasites that use host machinery to synthesize viral proteins. In infected eukaryotes, viral secreted and transmembrane proteins are synthesized at the endoplasmic reticulum (ER). Many viruses refashion ER membranes into bespoke factories where viral products accumulate while evading host pattern recognition receptors. ER processes are tightly regulated to maintain cellular homeostasis, so viruses must either conform to ER regulatory mechanisms or subvert them to ensure efficient viral replication. Reticulophagy is a catabolic process that directs lysosomal degradation of ER components. There is accumulating evidence that reticulophagy serves as a form of antiviral defense; we call this defense "xERophagy" to acknowledge its relationship to xenophagy, the catabolic degradation of microorganisms by macroautophagy/autophagy. In turn, viruses can subvert reticulophagy to suppress host antiviral responses and support efficient viral replication. Here, we review the evidence for functional interplay between viruses and the host reticulophagy machinery.

Keywords:  Autophagy; endoplasmic reticulum; reticulophagy; unfolded protein response; virus

DOI:  https://doi.org/10.1080/15548627.2024.2414424
Nat Commun. 2024 Oct 15. 15(1): 8895

Targeting IRE1α reprograms the tumor microenvironment and enhances anti-tumor immunity in prostate cancer.

Bilal Unal, Omer Faruk Kuzu, Yang Jin, Daniel Osorio, Wanja Kildal, Manohar Pradhan, Sonia H Y Kung, Htoo Zarni Oo, Mads Daugaard, Mikkel Vendelbo, John B Patterson, Martin Kristian Thomsen, Marieke Lydia Kuijjer, Fahri Saatcioglu.

Unfolded protein response (UPR) is a central stress response pathway that is hijacked by tumor cells for their survival. Here, we find that IRE1α signaling, one of the canonical UPR arms, is increased in prostate cancer (PCa) patient tumors. Genetic or small molecule inhibition of IRE1α in syngeneic mouse PCa models and an orthotopic model decreases tumor growth. IRE1α ablation in cancer cells potentiates interferon responses and activates immune system related pathways in the tumor microenvironment (TME). Single-cell RNA-sequencing analysis reveals that targeting IRE1α in cancer cells reduces tumor-associated macrophage abundance. Consistently, the small molecule IRE1α inhibitor MKC8866, currently in clinical trials, reprograms the TME and enhances anti-PD-1 therapy. Our findings show that IRE1α signaling not only promotes cancer cell growth and survival but also interferes with anti-tumor immunity in the TME. Thus, targeting IRE1α can be a promising approach for improving anti-PD-1 immunotherapy in PCa.

DOI: https://doi.org/10.1038/s41467-024-53039-1
Cell Rep. 2024 Oct 15. pii: S2211-1247(24)01215-4. [Epub ahead of print]43(11): 114864

Defective ribosome assembly impairs leukemia progression in a murine model of acute myeloid leukemia.

Daniel Sjövall, Sudip Ghosh, Narcis Fernandez-Fuentes, Talia Velasco-Hernandez, Anna Hogmalm, Pablo Menendez, Jenny Hansson, Carolina Guibentif, Pekka Jaako.

  Despite an advanced understanding of disease mechanisms, the current therapeutic regimen fails to cure most patients with acute myeloid leukemia (AML). In the present study, we address the role of ribosome assembly in leukemia cell function. We apply patient datasets and murine models to demonstrate that immature leukemia cells in mixed-lineage leukemia-rearranged AML are characterized by relatively high ribosome biogenesis and protein synthesis rates. Using a model with inducible regulation of ribosomal subunit joining, we show that defective ribosome assembly extends survival in mice with AML. Single-cell RNA sequencing and proteomic analyses reveal that leukemia cell adaptation to defective ribosome assembly is associated with an increase in ribosome biogenesis and deregulation of the transcription factor landscape. Finally, we demonstrate that defective ribosome assembly shows antileukemia efficacy in p53-deficient AML. Our study unveils the critical requirement of a high protein synthesis rate for leukemia progression and highlights ribosome assembly as a therapeutic target in AML.

Keywords:  AML; CP: Cancer; MLL; eIF6; leukemia; leukemia stem cell; mRNA translation; protein synthesis; ribosome; scRNA-seq

DOI:  https://doi.org/10.1016/j.celrep.2024.114864
Nat Chem Biol. 2024 Oct 16.

Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins.

Laurence J Seabrook, Carolina N Franco, Cody A Loy, Jaida Osman, Callie Fredlender, Jan Zimak, Melissa Campos, Steven T Nguyen, Richard L Watson, Samantha R Levine, Marian F Khalil, Kaelyn Sumigray, Darci J Trader, Lauren V Albrecht.

A paradigm shift in drug development is the discovery of small molecules that harness the ubiquitin-proteasomal pathway to eliminate pathogenic proteins. Here we provide a modality for targeted protein degradation in lysosomes. We exploit an endogenous lysosomal pathway whereby protein arginine methyltransferases (PRMTs) initiate substrate degradation via arginine methylation. We developed a heterobifunctional small molecule, methylarginine targeting chimera (MrTAC), that recruits PRMT1 to a target protein for induced degradation in lysosomes. MrTAC compounds degraded substrates across cell lines, timescales and doses. MrTAC degradation required target protein methylation for subsequent lysosomal delivery via microautophagy. A library of MrTAC molecules exemplified the generality of MrTAC to degrade known targets and neo-substrates-glycogen synthase kinase 3β, MYC, bromodomain-containing protein 4 and histone deacetylase 6. MrTAC selectively degraded target proteins and drove biological loss-of-function phenotypes in survival, transcription and proliferation. Collectively, MrTAC demonstrates the utility of endogenous lysosomal proteolysis in the generation of a new class of small molecule degraders.

DOI: https://doi.org/10.1038/s41589-024-01741-y
Nat Biotechnol. 2024 Oct 16.

Global profiling of protein complex dynamics with an experimental library of protein interaction markers.

Christian Dörig, Cathy Marulli, Thomas Peskett, Norbert Volkmar, Lorenzo Pantolini, Gabriel Studer, Camilla Paleari, Fabian Frommelt, Torsten Schwede, Natalie de Souza, Yves Barral, Paola Picotti.

Methods to systematically monitor protein complex dynamics are needed. We introduce serial ultrafiltration combined with limited proteolysis-coupled mass spectrometry (FLiP-MS), a structural proteomics workflow that generates a library of peptide markers specific to changes in PPIs by probing differences in protease susceptibility between complex-bound and monomeric forms of proteins. The library includes markers mapping to protein-binding interfaces and markers reporting on structural changes that accompany PPI changes. Integrating the marker library with LiP-MS data allows for global profiling of protein-protein interactions (PPIs) from unfractionated lysates. We apply FLiP-MS to Saccharomyces cerevisiae and probe changes in protein complex dynamics after DNA replication stress, identifying links between Spt-Ada-Gcn5 acetyltransferase activity and the assembly state of several complexes. FLiP-MS enables protein complex dynamics to be probed on any perturbation, proteome-wide, at high throughput, with peptide-level structural resolution and informing on occupancy of binding interfaces, thus providing both global and molecular views of a system under study.

DOI: https://doi.org/10.1038/s41587-024-02432-8
Nat Commun. 2024 Oct 14. 15(1): 8868

The genetic architecture of protein interaction affinity and specificity.

Alexandra M Bendel, Andre J Faure, Dominique Klein, Kenji Shimada, Romane Lyautey, Nicole Schiffelholz, Georg Kempf, Simone Cavadini, Ben Lehner, Guillaume Diss.

The encoding and evolution of specificity and affinity in protein-protein interactions is poorly understood. Here, we address this question by quantifying how all mutations in one protein, JUN, alter binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. We fit a global thermodynamic model to the data to reveal that most affinity changing mutations equally affect JUN's affinity to all its interaction partners. Mutations that alter binding specificity are relatively rare but distributed throughout the interaction interface. Specificity is determined both by features that promote on-target interactions and by those that prevent off-target interactions. Approximately half of the specificity-defining residues in JUN contribute both to promoting on-target binding and preventing off-target binding. Nearly all specificity-altering mutations in the interaction interface are pleiotropic, also altering affinity to all partners. In contrast, mutations outside the interface can tune global affinity without affecting specificity. Our results reveal the distributed encoding of specificity and affinity in an interaction interface and how coiled-coils provide an elegant solution to the challenge of optimizing both specificity and affinity in a large protein family.

DOI: https://doi.org/10.1038/s41467-024-53195-4
Cell. 2024 Oct 14. pii: S0092-8674(24)01090-0. [Epub ahead of print]

IRE1α silences dsRNA to prevent taxane-induced pyroptosis in triple-negative breast cancer.

Longyong Xu, Fanglue Peng, Qin Luo, Yao Ding, Fei Yuan, Liting Zheng, Wei He, Sophie S Zhang, Xin Fu, Jin Liu, Ayse Sena Mutlu, Shuyue Wang, Ralf Bernd Nehring, Xingyu Li, Qianzi Tang, Catherine Li, Xiangdong Lv, Lacey E Dobrolecki, Weijie Zhang, Dong Han, Na Zhao, Eric Jaehnig, Jingyi Wang, Weiche Wu, Davis A Graham, Yumei Li, Rui Chen, Weiyi Peng, Yiwen Chen, Andre Catic, Zhibin Zhang, Bing Zhang, Anthony M Mustoe, Albert C Koong, George Miles, Michael T Lewis, Meng C Wang, Susan M Rosenberg, Bert W O'Malley, Thomas F Westbrook, Han Xu, Xiang H-F Zhang, C Kent Osborne, Jin Billy Li, Matthew J Ellis, Mothaffar F Rimawi, Jeffrey M Rosen, Xi Chen.

  Chemotherapy is often combined with immune checkpoint inhibitor (ICIs) to enhance immunotherapy responses. Despite the approval of chemo-immunotherapy in multiple human cancers, many immunologically cold tumors remain unresponsive. The mechanisms determining the immunogenicity of chemotherapy are elusive. Here, we identify the ER stress sensor IRE1α as a critical checkpoint that restricts the immunostimulatory effects of taxane chemotherapy and prevents the innate immune recognition of immunologically cold triple-negative breast cancer (TNBC). IRE1α RNase silences taxane-induced double-stranded RNA (dsRNA) through regulated IRE1-dependent decay (RIDD) to prevent NLRP3 inflammasome-dependent pyroptosis. Inhibition of IRE1α in Trp53-/- TNBC allows taxane to induce extensive dsRNAs that are sensed by ZBP1, which in turn activates NLRP3-GSDMD-mediated pyroptosis. Consequently, IRE1α RNase inhibitor plus taxane converts PD-L1-negative, ICI-unresponsive TNBC tumors into PD-L1high immunogenic tumors that are hyper-sensitive to ICI. We reveal IRE1α as a cancer cell defense mechanism that prevents taxane-induced danger signal accumulation and pyroptotic cell death.

Keywords:  ER stress; IRE1α; PD-L1-negative breast cancer; dsRNA; pyroptosis

DOI:  https://doi.org/10.1016/j.cell.2024.09.032
J Mol Recognit. 2024 Oct 12. e3108

Toward Understanding the Mechanism of Client-Selective Small Molecule Inhibitors of the Sec61 Translocon.

Nidhi Sorout, Volkhard Helms.

The Sec61 translocon mediates the translocation of numerous, newly synthesized precursor proteins into the lumen of the endoplasmic reticulum or their integration into its membrane. Recently, structural biology revealed conformations of idle or substrate-engaged Sec61, and likewise its interactions with the accessory membrane proteins Sec62, Sec63, and TRAP, respectively. Several natural and synthetic small molecules have been shown to block Sec61-mediated protein translocation. Since this is a key step in protein biogenesis, broad inhibition is generally cytotoxic, which may be problematic for a putative drug target. Interestingly, several compounds exhibit client-selective modes of action, such that only translocation of certain precursor proteins was affected. Here, we discuss recent advances of structural biology, molecular modelling, and molecular screening that aim to use Sec61 as feasible drug target.

DOI: https://doi.org/10.1002/jmr.3108
Database (Oxford). 2024 Oct 12. pii: baae111. [Epub ahead of print]2024

ELiAH: the atlas of E3 ligases in human tissues for targeted protein degradation with reduced off-target effect.

Hyojung Paik, Chunryong Oh, Sajid Hussain, Sangjae Seo, Soon Woo Park, Tae Lyun Ko, Ari Lee.

The development of therapeutic agents has mainly focused on designing small molecules to modulate target proteins or genes which are conventionally druggable. Therefore, targeted protein degradation (TPD) for undruggable cases has emerged as promising pharmaceutical approach. TPD, often referred PROTACs (PROteolysis TArgeting Chimeras), uses a linker to degrade target proteins by hijacking the ubiquitination system. Therefore, unravel the relationship including reversal and co-expression between E3 ligands and other possible target genes in various human tissues is essential to mitigate off-target effects of TPD. Here, we developed the atlas of E3 ligases in human tissues (ELiAH), to prioritize E3 ligase-target gene pairs for TPD. Leveraging over 2900 of RNA-seq profiles consisting of 11 human tissues from the GTEx (genotype-tissue expression) consortium, users of ELiAH can identify tissue-specific genes and E3 ligases (FDR P-value of Mann-Whitney test < .05). ELiAH unravels 933 830 relationships consisting of 614 E3 ligases and 20 924 of expressed genes considering degree of tissue specificity, which are indispensable for ubiquitination based TPD development. In addition, docking properties of those relationships are also modeled using RosettaDock. Therefore, ELiAH presents comprehensive repertoire of E3 ligases for ubiquitination-based TPD drug development avoiding off-target effects. Database URL: https://eliahdb.org.

DOI: https://doi.org/10.1093/database/baae111
Mol Cell. 2024 Oct 14. pii: S1097-2765(24)00782-2. [Epub ahead of print]

Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma.

Sadaaki Nishimura, Juan F Linares, Antoine L'Hermitte, Angeles Duran, Tania Cid-Diaz, Anxo Martinez-Ordoñez, Marc Ruiz-Martinez, Yotaro Kudo, Antonio Marzio, Mathias Heikenwalder, Lewis R Roberts, Maria T Diaz-Meco, Jorge Moscat.

  Hepatocellular carcinoma (HCC) emerges from chronic inflammation, to which activation of hepatic stellate cells (HSCs) contributes by shaping a pro-tumorigenic microenvironment. Key to this process is p62, whose inactivation leads to enhanced hepatocarcinogenesis. Here, we show that p62 activates the interferon (IFN) cascade by promoting STING ubiquitination by tripartite motif protein 32 (TRIM32) in HSCs. p62, binding neighbor of BRCA1 gene 1 (NBR1) and STING, triggers the IFN cascade by displacing NBR1, which normally prevents the interaction of TRIM32 with STING and its subsequent activation. Furthermore, NBR1 also antagonizes STING by promoting its trafficking to the endosome-lysosomal compartment for degradation independent of autophagy. Of functional relevance, NBR1 deletion completely reverts the tumor-promoting function of p62-deficient HSCs by rescuing the inhibited STING-IFN pathway, thus enhancing anti-tumor responses mediated by CD8+ T cells. Therefore, NBR1 emerges as a synthetic vulnerability of p62 deficiency in HSCs by promoting the STING/IFN pathway, which boosts anti-tumor CD8+ T cell responses to restrain HCC progression.

Keywords:  CD8(+) T cells; NBR1; STING; TRIM32; hepatic stellate cells; hepatocellular carcinoma; interferon; microenvironment; p62

DOI:  https://doi.org/10.1016/j.molcel.2024.09.026
Mol Oncol. 2024 Oct 17.

The translatome of glioblastoma.

Fleur M G Cornelissen, Zhaoren He, Edward Ciputra, Richard R de Haas, Ammarina Beumer-Chuwonpad, David Noske, W Peter Vandertop, Sander R Piersma, Connie R Jiménez, Cornelis Murre, Bart A Westerman.

  Glioblastoma (GB), the most common and aggressive brain tumor, demonstrates intrinsic resistance to current therapies, resulting in poor clinical outcomes. Cancer progression can be partially attributed to the deregulation of protein translation mechanisms that drive cancer cell growth. In this study, we present the translatome landscape of GB as a valuable data resource. Eight patient-derived GB sphere cultures (GSCs) were analyzed using ribosome profiling and messenger RNA (mRNA) sequencing. We investigated inter-cell-line differences through differential expression analysis at both the translatome and transcriptome levels. Translational changes post-radiotherapy were assessed at 30 and 60 min. The translation of non-coding RNAs (ncRNAs) was validated using in-house and public mass spectrometry (MS) data, whereas RNA expression was confirmed by quantitative PCR (qPCR). Our findings demonstrate that ribosome sequencing provides more detailed information than MS or transcriptional analyses. Transcriptional similarities among GSCs correlate with translational similarities, aligning with previously defined subtypes such as proneural and mesenchymal. Additionally, we identified a broad spectrum of open reading frame types in both coding and non-coding mRNA regions, including long non-coding RNAs (lncRNAs) and pseudogenes undergoing active translation. Translation of ncRNAs into peptides was independently confirmed by in-house data and external MS data. We also observed that translational regulation of histones (downregulated) and splicing factors (upregulated) occurs in response to radiotherapy. These data offer new insights into genome-wide protein synthesis, identifying translationally regulated genes and alternative translation initiation sites in GB under normal and radiotherapeutic conditions, providing a rich resource for GB research. Further functional validation of differentially expressed genes after radiotherapy is needed. Understanding translational control in GB can reveal mechanistic insights and identify currently unknown biomarkers, ultimately enhancing the diagnosis and treatment of this aggressive brain cancer.

Keywords:  glioblastoma; non‐coding RNA; radioresistance; radiotherapy; translatome

DOI:  https://doi.org/10.1002/1878-0261.13743
Bio Protoc. 2024 Oct 05. 14(19): e5080

Fluorescence Lifetime-Assisted Probing of Protein Aggregation with sub-Organellar Resolution.

Karnika Gupta, Daniel C Maddison, Eduardo P Melo, Ana Rosa M da Costa, Edward Avezov.

  Protein misfolding fuels multiple neurodegenerative diseases, but existing techniques lack the resolution to pinpoint the location and physical properties of aggregates within living cells. Our protocol describes high-resolution confocal and fluorescent lifetime microscopy (Fast 3D FLIM) of an aggregation probing system. This system involves a metastable HaloTag protein (HT-aggr) labeled with P1 solvatochromic fluorophore, which can be targeted to subcellular compartments. This strategy allows to distinguish between aggregated and folded probe species, since P1 fluorophore changes its lifetime depending on the hydrophobicity of its microenvironment. The probe is not fluorescence intensity-dependent, overcoming issues related to intensity-based measurements of labeled proteins, such as control of probe quantity due to differences in expression or photobleaching of a proportion of the fluorophore population. Our approach reports on the performance of the machinery dealing with aggregation-prone substrates and thus opens doors to studying proteostasis and its role in neurodegenerative diseases. Key features • Aggregation state: Tracks aggregate formation and disaggregation with pulse-chase experiments • Sub-organellar resolution: Pinpoints and allows control of aggregate location within the cell, exceeding traditional techniques • Quantitative analysis: Measures aggregate load through image analysis • Methodology: • Metastable HaloTag variant labeling with a solvatochromic small-molecule reporter ligand • High-resolution confocal microscopy coupled with FLIM for aggregate identification and localization • Image analysis for aggregate quantification and distribution within the ER • Pulse-chase experiments to track aggregates.

Keywords:  3D-FLIM; Disaggregation; ER; FLIM; HT-aggrER

DOI:  https://doi.org/10.21769/BioProtoc.5080
Genome Biol. 2024 Oct 14. 25(1): 268

The ribosome profiling landscape of yeast reveals a high diversity in pervasive translation.

Chris Papadopoulos, Hugo Arbes, David Cornu, Nicolas Chevrollier, Sandra Blanchet, Paul Roginski, Camille Rabier, Safiya Atia, Olivier Lespinet, Olivier Namy, Anne Lopes.

  BACKGROUND: Pervasive translation is a widespread phenomenon that plays a critical role in the emergence of novel microproteins, but the diversity of translation patterns contributing to their generation remains unclear. Based on 54 ribosome profiling (Ribo-Seq) datasets, we investigated the yeast Ribo-Seq landscape using a representation framework that allows the comprehensive inventory and classification of the entire diversity of Ribo-Seq signals, including non-canonical ones.RESULTS: We show that if coding regions occupy specific areas of the Ribo-Seq landscape, noncoding regions encompass a wide diversity of Ribo-Seq signals and, conversely, populate the entire landscape. Our results show that pervasive translation can, nevertheless, be associated with high specificity, with 1055 noncoding ORFs exhibiting canonical Ribo-Seq signals. Using mass spectrometry under standard conditions or proteasome inhibition with an in-house analysis protocol, we report 239 microproteins originating from noncoding ORFs that display canonical but also non-canonical Ribo-Seq signals. Each condition yields dozens of additional microprotein candidates with comparable translation properties, suggesting a larger population of volatile microproteins that are challenging to detect. Our findings suggest that non-canonical translation signals may harbor valuable information and underscore the significance of considering them in proteogenomic studies. Finally, we show that the translation outcome of a noncoding ORF is primarily determined by the initiating codon and the codon distribution in its two alternative frames, rather than features indicative of functionality.
CONCLUSION: Our results enable us to propose a topology of a species' Ribo-Seq landscape, opening the way to comparative analyses of this translation landscape under different conditions.

Keywords:  De novo coding products; Genome evolution; Non-canonical translation signals; Noncoding genome; Pervasive translation

DOI:  https://doi.org/10.1186/s13059-024-03403-7
Nature. 2024 Oct 16.

Spatial proteomics identifies JAKi as treatment for a lethal skin disease.

Thierry M Nordmann, Holly Anderton, Akito Hasegawa, Lisa Schweizer, Peng Zhang, Pia-Charlotte Stadler, Ankit Sinha, Andreas Metousis, Florian A Rosenberger, Maximilian Zwiebel, Takashi K Satoh, Florian Anzengruber, Maximilian T Strauss, Maria C Tanzer, Yuki Saito, Ting Gong, Marvin Thielert, Haruna Kimura, Natasha Silke, Edwin H Rodriguez, Gaetana Restivo, Hong Ha Nguyen, Annette Gross, Laurence Feldmeyer, Lukas Joerg, Mitchell P Levesque, Peter J Murray, Saskia Ingen-Housz-Oro, Andreas Mund, Riichiro Abe, John Silke, Chao Ji, Lars E French, Matthias Mann.

Toxic epidermal necrolysis (TEN) is a fatal drug-induced skin reaction triggered by common medications and is an emerging public health issue1-3. Patients with TEN undergo severe and sudden epidermal detachment caused by keratinocyte cell death. Although molecular mechanisms that drive keratinocyte cell death have been proposed, the main drivers remain unknown, and there is no effective therapy for TEN4-6. Here, to systematically map molecular changes that are associated with TEN and identify potential druggable targets, we utilized deep visual proteomics, which provides single-cell-based, cell-type-resolution proteomics7,8. We analysed formalin-fixed, paraffin-embedded archived skin tissue biopsies of three types of cutaneous drug reactions with varying severity and quantified more than 5,000 proteins in keratinocytes and skin-infiltrating immune cells. This revealed a marked enrichment of type I and type II interferon signatures in the immune cell and keratinocyte compartment of patients with TEN, as well as phosphorylated STAT1 activation. Targeted inhibition with the pan-JAK inhibitor tofacitinib in vitro reduced keratinocyte-directed cytotoxicity. In vivo oral administration of tofacitinib, baricitinib or the JAK1-specific inhibitors abrocitinib or upadacitinib ameliorated clinical and histological disease severity in two distinct mouse models of TEN. Crucially, treatment with JAK inhibitors (JAKi) was safe and associated with rapid cutaneous re-epithelialization and recovery in seven patients with TEN. This study uncovers the JAK/STAT and interferon signalling pathways as key pathogenic drivers of TEN and demonstrates the potential of targeted JAKi as a curative therapy.

DOI: https://doi.org/10.1038/s41586-024-08061-0