bims-proteo 2024-09-15 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–09–15
thirty-six papers selected by
Eric Chevet, INSERM

NEMF-mediated CAT-tailing defines distinct branches of translocation-associated quality control.
Cytosolic FKBPL and ER-resident CKAP4 co-regulates ER-phagy and protein secretion.
Targeted proteomics addresses selectivity and complexity of protein degradation by autophagy.
Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery.
An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.
The prolyl isomerase FKBP11 is a secretory translocon accessory factor.
FicD sensitizes cellular response to glucose fluctuations in mouse embryonic fibroblasts.
Extrapolating Lessons from Targeted Protein Degradation to Other Proximity-Inducing Drugs.
Catchers of folding gone awry: a tale of small heat shock proteins.
Ubiquitin-driven protein condensation stabilizes clathrin-mediated endocytosis.
Comprehensive detection and characterization of human druggable pockets through binding site descriptors.
Ubiquitylation of nucleic acids by DELTEX ubiquitin E3 ligase DTX3L.
Increased RNA and protein degradation is required for counteracting transcriptional burden and proteotoxic stress in human aneuploid cells.
Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage.
FBXO38 is dispensable for PD-1 regulation.
IRE1α-XBP1s axis regulates SREBP1-dependent MRP1 expression to promote chemoresistance in non-small cell lung cancer cells.
Cellular processing of beneficial de novo emerging proteins.
NEDD4 family ubiquitin ligase AIP4 interacts with Alix to enable HBV naked capsid egress in an Alix ubiquitination-independent manner.
TEX264 drives selective autophagy of DNA lesions to promote DNA repair and cell survival.
CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction.
Pooled endogenous protein tagging and recruitment for systematic profiling of protein function.
RING1 missense variants reveal sensitivity of DNA damage repair to H2A monoubiquitination dosage during neurogenesis.
DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment.
Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators.
Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis.
Phosphorylation of tau at a single residue inhibits binding to the E3 ubiquitin ligase, CHIP.
HOPping to the vacuole: Autophagosome and late endosomes combine to control plant autophagosome degradation.
RAPIDASH: Tag-free enrichment of ribosome-associated proteins reveals composition dynamics in embryonic tissue, cancer cells, and macrophages.
Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease.
Structural roles of Ump1 and β-subunit propeptides in proteasome biogenesis.
Inhibition of nucleo-cytoplasmic proteasome translocation by the aromatic amino acids or silencing Sestrin3-their sensing mediator-is tumor suppressive.
Cannabinoid combination targets NOTCH1-mutated T-cell acute lymphoblastic leukemia through the integrated stress response pathway.
Antibiotics damage the colonic mucus barrier in a microbiota-independent manner.
Proteasomes safeguard the plant stress granule homeostasis.
Automated design of multi-target ligands by generative deep learning.
The fitness cost of spurious phosphorylation.

bioRxiv. 2024 Aug 28. pii: 2024.08.27.610005. [Epub ahead of print]

NEMF-mediated CAT-tailing defines distinct branches of translocation-associated quality control.

Amanda Ennis, Lihui Wang, Xiaorong Wang, Clinton Yu, Layla Saidi, Yue Xu, Sijung Yun, Lan Huang, Yihong Ye.

Ribosome stalling during co-translational translocation at the endoplasmic reticulum (ER) causes translocon clogging and impairs ER protein biogenesis. Mammalian cells resolve translocon clogging vial a poorly characterized translocation-associated quality control (TAQC) process. Here, we combine genome-wide CRISPR screen with live cell imaging to dissect the molecular linchpin of TAQC. We show that substrates translated from mRNAs bearing a ribosome stalling poly(A) sequence are degraded by lysosomes and the proteasome, while substrates encoded by non-stop mRNAs are degraded by an unconventional ER-associated degradation (ERAD) mechanism involving ER-to-Golgi trafficking and KDEL-dependent substrate retrieval. The triaging diversity appears to result from the heterogeneity of NEMF-mediated CATylation, because a systematic characterization of representative CAT-tail mimetics establishes an AT-rich tail as a "degron" for ERAD, whereas an AG-rich tail can direct a secretory protein to the lysosome. Our study reveals an unexpected protein sorting function for CAT-tailing that safeguards ER protein biogenesis.

DOI: https://doi.org/10.1101/2024.08.27.610005
Nat Commun. 2024 Sep 09. 15(1): 7886

Cytosolic FKBPL and ER-resident CKAP4 co-regulates ER-phagy and protein secretion.

Cathena Meiling Li, Jaemin Kang, Jongyeon Baek, Youbin Kim, Heemin Park, Yong-Keun Jung.

Endoplasmic reticulum quality control is crucial for maintaining cellular homeostasis and adapting to stress conditions. Although several ER-phagy receptors have been identified, the collaboration between cytosolic and ER-resident factors in ER fragmentation and ER-phagy regulation remains unclear. Here, we perform a phenotype-based gain-of-function screen and identify a cytosolic protein, FKBPL, functioning as an ER-phagy regulator. Overexpression of FKBPL triggers ER fragmentation and ER-phagy. FKBPL has multiple protein binding domains, can self-associate and might act as a scaffold connecting CKAP4 and LC3/GABARAPs. CKAP4 serves as a bridge between FKBPL and ER-phagy cargo. ER-phagy-inducing conditions increase FKBPL-CKAP4 interaction followed by FKBPL oligomerization at the ER, leading to ER-phagy. In addition, FKBPL-CKAP4 deficiency leads to Golgi disassembly and lysosome impairment, and an increase in ER-derived secretory vesicles and enhances cytosolic protein secretion via microvesicle shedding. Taken together, FKBPL with the aid of CKAP4 induces ER fragmentation and ER-phagy, and FKBPL-CKAP4 deficiency facilitates protein secretion.

DOI: https://doi.org/10.1038/s41467-024-52188-7
Autophagy. 2024 Sep 08.

Targeted proteomics addresses selectivity and complexity of protein degradation by autophagy.

Alexandre Leytens, Rocío Benítez-Fernández, Carlos Jiménez-García, Carole Roubaty, Michael Stumpe, Patricia Boya, Jörn Dengjel.

  Macroautophagy/autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require de novo protein synthesis. Historically, autophagy has been regarded as non-selective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, i.e. selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-related proteins covering process-relevant proteins such as the initiation complex and the Atg8-family protein lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy-inducing conditions in contrast to SARs, in a cell-line dependent manner. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and -independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy flux.

Keywords:  ER-phagy; mass spectrometry; mitophagy; parallel reaction monitoring; reticulophagy; selective autophagy receptors

DOI:  https://doi.org/10.1080/15548627.2024.2396792
bioRxiv. 2024 Aug 28. pii: 2024.08.28.609967. [Epub ahead of print]

Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery.

Elias Adriaenssens, Stefan Schaar, Annan S I Cook, Jan F M Stuke, Justyna Sawa-Makarska, Thanh Ngoc Nguyen, Xuefeng Ren, Martina Schuschnig, Julia Romanov, Grace Khuu, Michael Lazarou, Gerhard Hummer, James H Hurley, Sascha Martens.

Selective autophagy is a lysosomal degradation pathway that is critical for maintaining cellular homeostasis by disposing of harmful cellular material. While the mechanisms by which soluble cargo receptors recruit the autophagy machinery are becoming increasingly clear, the principles governing how organelle-localized transmembrane cargo receptors initiate selective autophagy remain poorly understood. Here, we demonstrate that transmembrane cargo receptors can initiate autophagosome biogenesis not only by recruiting the upstream FIP200/ULK1 complex but also via a WIPI-ATG13 complex. This latter pathway is employed by the BNIP3/NIX receptors to trigger mitophagy. Additionally, other transmembrane mitophagy receptors, including FUNDC1 and BCL2L13, exclusively use the FIP200/ULK1 complex, while FKBP8 and the ER-phagy receptor TEX264 are capable of utilizing both pathways to initiate autophagy. Our study defines the molecular rules for initiation by transmembrane cargo receptors, revealing remarkable flexibility in the assembly and activation of the autophagy machinery, with significant implications for therapeutic interventions.

DOI: https://doi.org/10.1101/2024.08.28.609967
Cells. 2024 Sep 06. pii: 1498. [Epub ahead of print]13(17):

An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.

Daniel Wolf, Chiara Röder, Michael Sendtner, Patrick Lüningschrör.

  ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.

Keywords:  ER stress; ER-phagy; UPR; calnexin; unfolded protein response

DOI:  https://doi.org/10.3390/cells13171498
Mol Biol Cell. 2024 Sep 11. mbcE24070305

The prolyl isomerase FKBP11 is a secretory translocon accessory factor.

Amanda DiGuilio, Ben Cheng, Frank Zhong, Roshan Jha, Yu Wan, Andrei Anghel, Hong Hu, Evgenia Shishkova, Zhe Ji, Joshua J Coon, Robert J Keenan.

Eukaryotic cells encode thousands of secretory and membrane proteins, many of which are cotranslationally translocated into or across the endoplasmic reticulum (ER). Nascent polypeptides entering the ER encounter a network of molecular chaperones and enzymes that facilitate their folding. A rate-limiting step for some proteins is the trans-to-cis isomerization of the peptide bond between proline and the residue preceding it. The human ER contains six prolyl isomerases, but the function, organization and substrate range of these proteins is not clear. Here we show that the metazoan-specific, prolyl isomerase FKBP11 binds to ribosome-translocon complexes (RTCs) in the ER membrane, dependent on its single transmembrane domain (TMD) and a conserved, positively charged region at its cytosolic C-terminus. High throughput mRNA sequencing shows selective engagement with ribosomes synthesizing secretory and membrane proteins with long translocated segments, and functional analysis shows reduced stability of two such proteins, EpCAM and PTTG1IP, in cells depleted of FKBP11. We propose that FKBP11 is a translocon accessory factor that acts on a broad range of soluble secretory and transmembrane proteins during their synthesis at the ER.

DOI: https://doi.org/10.1091/mbc.E24-07-0305
Proc Natl Acad Sci U S A. 2024 Sep 17. 121(38): e2400781121

FicD sensitizes cellular response to glucose fluctuations in mouse embryonic fibroblasts.

Burak Gulen, Aubrie Blevins, Lisa N Kinch, Kelly A Servage, Nathan M Stewart, Hillery F Gray, Amanda K Casey, Kim Orth.

  During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis.

Keywords:  AMPylation; BiP; ER stress; Fic enzymes posttranslational modifications; unfolded protein response

DOI:  https://doi.org/10.1073/pnas.2400781121
ACS Chem Biol. 2024 Sep 12.

Extrapolating Lessons from Targeted Protein Degradation to Other Proximity-Inducing Drugs.

Georg E Winter.

Targeted protein degradation (TPD) is an emerging pharmacologic strategy. It relies on small-molecule "degraders" that induce proximity of a component of an E3 ubiquitin ligase complex and a target protein to induce target ubiquitination and subsequent proteasomal degradation. Essentially, degraders thus expand the function of E3 ligases, allowing them to degrade proteins they would not recognize in the absence of the small molecule. Over the past decade, insights gained from identifying, designing, and characterizing various degraders have significantly enhanced our understanding of TPD mechanisms, precipitating in rational degrader discovery strategies. In this Account, I aim to explore how these insights can be extrapolated to anticipate both opportunities and challenges of utilizing the overarching concept of proximity-inducing pharmacology to manipulate other cellular circuits for the dissection of biological mechanisms and for therapeutic purposes.

DOI: https://doi.org/10.1021/acschembio.4c00191
Trends Biochem Sci. 2024 Sep 12. pii: S0968-0004(24)00190-7. [Epub ahead of print]

Catchers of folding gone awry: a tale of small heat shock proteins.

Carsten Peters, Martin Haslbeck, Johannes Buchner.

  Small heat shock proteins (sHsps) are an important part of the cellular system maintaining protein homeostasis under physiological and stress conditions. As molecular chaperones, they form complexes with different non-native proteins in an ATP-independent manner. Many sHsps populate ensembles of energetically similar but different-sized oligomers. Regulation of chaperone activity occurs by changing the equilibrium of these ensembles. This makes sHsps a versatile and adaptive system for trapping non-native proteins in complexes, allowing recycling with the help of ATP-dependent chaperones. In this review, we discuss progress in our understanding of the structural principles of sHsp oligomers and their functional principles, as well as their roles in aging and eye lens transparency.

Keywords:  molecular chaperones; protein aggregation; protein folding; protein homeostasis; small heat shock proteins; α-crystallin

DOI:  https://doi.org/10.1016/j.tibs.2024.08.003
PNAS Nexus. 2024 Sep;3(9): pgae342

Ubiquitin-driven protein condensation stabilizes clathrin-mediated endocytosis.

Feng Yuan, Sadhana Gollapudi, Kasey J Day, Grant Ashby, Arjun Sangani, Brandon T Malady, Liping Wang, Eileen M Lafer, Jon M Huibregtse, Jeanne C Stachowiak.

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple groups have reported that early endocytic proteins form flexible condensates, which provide a platform for efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic assemblies. In live-cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live-cell settings, dynamic exchange of Eps15 proteins, a measure of protein network stability, was decreased by Eps15-ubiquitin interactions and increased by loss of ubiquitin. These results collectively suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic internalization.

DOI: https://doi.org/10.1093/pnasnexus/pgae342
Nat Commun. 2024 Sep 10. 15(1): 7917

Comprehensive detection and characterization of human druggable pockets through binding site descriptors.

Arnau Comajuncosa-Creus, Guillem Jorba, Xavier Barril, Patrick Aloy.

Druggable pockets are protein regions that have the ability to bind organic small molecules, and their characterization is essential in target-based drug discovery. However, deriving pocket descriptors is challenging and existing strategies are often limited in applicability. We introduce PocketVec, an approach to generate pocket descriptors via inverse virtual screening of lead-like molecules. PocketVec performs comparably to leading methodologies while addressing key limitations. Additionally, we systematically search for druggable pockets in the human proteome, using experimentally determined structures and AlphaFold2 models, identifying over 32,000 binding sites across 20,000 protein domains. We then generate PocketVec descriptors for each site and conduct an extensive similarity search, exploring over 1.2 billion pairwise comparisons. Our results reveal druggable pocket similarities not detected by structure- or sequence-based methods, uncovering clusters of similar pockets in proteins lacking crystallized inhibitors and opening the door to strategies for prioritizing chemical probe development to explore the druggable space.

DOI: https://doi.org/10.1038/s41467-024-52146-3
EMBO Rep. 2024 Sep 06.

Ubiquitylation of nucleic acids by DELTEX ubiquitin E3 ligase DTX3L.

Kang Zhu, Chatrin Chatrin, Marcin J Suskiewicz, Vincent Aucagne, Benjamin Foster, Benedikt M Kessler, Ian Gibbs-Seymour, Dragana Ahel, Ivan Ahel.

  The recent discovery of non-proteinaceous ubiquitylation substrates broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated via ester bond formation. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is shared with DTX3, but not with the other DELTEX family members DTX1, DTX2 and DTX4. DTX3L shows preference for the 3'-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2, JOSD1 and SARS-CoV-2 PLpro. Overall, our study proposes reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications.

Keywords:  DELTEX; DTX3L; Genome Stability; Nucleic Acid Modification; Ubiquitin

DOI:  https://doi.org/10.1038/s44319-024-00235-1
Cancer Discov. 2024 Sep 09.

Increased RNA and protein degradation is required for counteracting transcriptional burden and proteotoxic stress in human aneuploid cells.

Marica Rosaria Ippolito, Johanna Zerbib, Yonatan Eliezer, Eli Reuveni, Sonia Vigano, Giuseppina De Feudis, Eldad D Shulman, Anouk Savir Kadmon, Rachel Slutsky, Tiangen Chang, Emma M Campagnolo, Silvia Taglietti, Simone Scorzoni, Sara Gianotti, Sara Martin, Julia Muenzner, Michael Mulleder, Nir Rozenblum, Carmela Rubolino, Tal Ben-Yishay, Kathrin Laue, Yael Cohen-Sharir, Ilaria Vigorito, Francesco Nicassio, Eytan Ruppin, Markus Ralser, Francisca Vazquez, Stefano Santaguida, Uri Ben-David.

Aneuploidy results in a stoichiometric imbalance of protein complexes that jeopardizes cellular fitness. Aneuploid cells thus need to compensate for the imbalanced DNA levels by regulating their RNA and protein levels, but the underlying molecular mechanisms remain unknown. Here, we dissected multiple diploid vs. aneuploid cell models. We found that aneuploid cells cope with transcriptional burden by increasing several RNA degradation pathways, and are consequently more sensitive to the perturbation of RNA degradation. At the protein level, aneuploid cells mitigate proteotoxic stress by reducing protein translation and increasing protein degradation, rendering them more sensitive to proteasome inhibition. These findings were recapitulated across hundreds of human cancer cell lines and primary tumors, and aneuploidy levels were significantly associated with the response of multiple myeloma patients to proteasome inhibitors. Aneuploid cells are therefore preferentially dependent on several key nodes along the gene expression process, creating clinically-actionable vulnerabilities in aneuploid cells.

DOI: https://doi.org/10.1158/2159-8290.CD-23-0309
Nat Commun. 2024 Sep 09. 15(1): 7772

Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage.

Johanna Zerbib, Marica Rosaria Ippolito, Yonatan Eliezer, Giuseppina De Feudis, Eli Reuveni, Anouk Savir Kadmon, Sara Martin, Sonia Viganò, Gil Leor, James Berstler, Julia Muenzner, Michael Mülleder, Emma M Campagnolo, Eldad D Shulman, Tiangen Chang, Carmela Rubolino, Kathrin Laue, Yael Cohen-Sharir, Simone Scorzoni, Silvia Taglietti, Alice Ratti, Chani Stossel, Talia Golan, Francesco Nicassio, Eytan Ruppin, Markus Ralser, Francisca Vazquez, Uri Ben-David, Stefano Santaguida.

Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induce chromosome mis-segregation in non-transformed RPE1-hTERT cells and derive multiple stable clones with various degrees of aneuploidy. We perform a systematic genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogate their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activate the DNA damage response and are more resistant to further DNA damage induction. Aneuploid cells also exhibit elevated RAF/MEK/ERK pathway activity and are more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitize aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. We validate these results in human cancer cell lines. Moreover, resistance of cancer patients to olaparib is associated with high levels of RAF/MEK/ERK signaling, specifically in highly-aneuploid tumors. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a therapeutically-relevant cellular dependency of aneuploid cells.

DOI: https://doi.org/10.1038/s41467-024-52176-x
EMBO Rep. 2024 Sep 12.

FBXO38 is dispensable for PD-1 regulation.

Nikol Dibus, Eva Salyova, Karolina Kolarova, Alikhan Abdirov, Michele Pagano, Ondrej Stepanek, Lukas Cermak.

  SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of protein substrates. The direct substrate recognition relies on a large family of F-box-domain-containing subunits. One of these substrate receptors is FBXO38, which is encoded by a gene found mutated in families with early-onset distal motor neuronopathy. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B. Loss of FBXO38 in mice results in growth retardation and defects in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and compromised centromere integrity. Moreover, it was reported that SCFFBXO38 mediates the degradation of PD-1, a key immune-checkpoint inhibitor in T cells. Here, we have re-addressed the link between SCFFBXO38 and PD-1 proteolysis. Our data do not support the notion that SCFFBXO38 directly or indirectly controls the abundance and stability of PD-1 in T cells.

Keywords:  Cullin; FBXO38; Immune Checkpoint; PD-1; Protein Degradation

DOI:  https://doi.org/10.1038/s44319-024-00220-8
Thorac Cancer. 2024 Sep 08.

IRE1α-XBP1s axis regulates SREBP1-dependent MRP1 expression to promote chemoresistance in non-small cell lung cancer cells.

Yuzhou Xu, Feng Gui, Zhe Zhang, Zhongyang Chen, Tiange Zhang, Yunhan Hu, Huijun Wei, Yuchen Fu, Xinde Chen, Zhihao Wu.

   BACKGROUND: Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and mediates an essential arm of the unfolded protein response (UPR). IRE1 reduces ER stress by upregulating the expression of multiple ER chaperones through activation of X-box-binding protein 1 (XBP1). Emerging lines of evidence have revealed that IRE1-XBP1 axis serves as a multipurpose signal transducer during oncogenic transformation and cancer development. In this study, we explore how IRE1-XBP1 signaling promotes chemoresistance in lung cancer.
METHODS: The expression patterns of UPR components and MRP1 were examined by Western blot. qRT-PCR was employed to determine RNA expression. The promoter activity was determined by luciferase reporter assay. Chemoresistant cancer cells were analyzed by viability, apoptosis. CUT & Tag (Cleavage under targets and tagmentation)-qPCR analysis was used for analysis of DNA-protein interaction.
RESULTS: Here we show that activation of IRE1α-XBP1 pathway leads to an increase in MDR-related protein 1 (MRP1) expression, which facilitates drug extrusion and confers resistance to cytotoxic chemotherapy. At the molecular level, XBP1-induced c-Myc is necessary for SREBP1 expression, and SREBP1 binds to the MRP1 promoter to directly regulate its transcription.
CONCLUSIONS: We conclude that IRE1α-XBP1 had important role in chemoresistance and appears to be a novel prognostic marker for lung cancer.

Keywords:  IRE1α; MRP1; SREBP1; XBP1; chemoresistance

DOI:  https://doi.org/10.1111/1759-7714.15442
bioRxiv. 2024 Aug 29. pii: 2024.08.28.610198. [Epub ahead of print]

Cellular processing of beneficial de novo emerging proteins.

Carly J Houghton, Nelson Castilho Coelho, Annette Chiang, Stefanie Hedayati, Saurin B Parikh, Nejla Ozbaki-Yagan, Aaron Wacholder, John Iannotta, Alexis Berger, Anne-Ruxandra Carvunis, Allyson F O'Donnell.

Novel proteins can originate de novo from non-coding DNA and contribute to species-specific adaptations. It is challenging to conceive how de novo emerging proteins may integrate pre-existing cellular systems to bring about beneficial traits, given that their sequences are previously unseen by the cell. To address this apparent paradox, we investigated 26 de novo emerging proteins previously associated with growth benefits in yeast. Microscopy revealed that these beneficial emerging proteins preferentially localize to the endoplasmic reticulum (ER). Sequence and structure analyses uncovered a common protein organization among all ER-localizing beneficial emerging proteins, characterized by a short hydrophobic C-terminus immediately preceded by a transmembrane domain. Using genetic and biochemical approaches, we showed that ER localization of beneficial emerging proteins requires the GET and SND pathways, both of which are evolutionarily conserved and known to recognize transmembrane domains to promote post-translational ER insertion. The abundance of ER-localizing beneficial emerging proteins was regulated by conserved proteasome- and vacuole-dependent processes, through mechanisms that appear to be facilitated by the emerging proteins' C-termini. Consequently, we propose that evolutionarily conserved pathways can convergently govern the cellular processing of de novo emerging proteins with unique sequences, likely owing to common underlying protein organization patterns.

DOI: https://doi.org/10.1101/2024.08.28.610198
PLoS Pathog. 2024 Sep;20(9): e1012485

NEDD4 family ubiquitin ligase AIP4 interacts with Alix to enable HBV naked capsid egress in an Alix ubiquitination-independent manner.

Sheng Shen, Dawei Cai, Hongyan Liang, Ge Zeng, Wendong Liu, Ran Yan, Xiaoyang Yu, Hu Zhang, Shi Liu, Wanying Li, Rui Deng, Xingyu Lu, Yuanjie Liu, Jian Sun, Haitao Guo.

Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.

DOI: https://doi.org/10.1371/journal.ppat.1012485
Cell. 2024 Sep 05. pii: S0092-8674(24)00911-5. [Epub ahead of print]

TEX264 drives selective autophagy of DNA lesions to promote DNA repair and cell survival.

Pauline Lascaux, Gwendoline Hoslett, Sara Tribble, Camilla Trugenberger, Ivan Antičević, Cecile Otten, Ignacio Torrecilla, Stelios Koukouravas, Yichen Zhao, Hongbin Yang, Ftoon Aljarbou, Annamaria Ruggiano, Wei Song, Cristiano Peron, Giulio Deangeli, Enric Domingo, James Bancroft, Loïc Carrique, Errin Johnson, Iolanda Vendrell, Roman Fischer, Alvin Wei Tian Ng, Joanne Ngeow, Vincenzo D'Angiolella, Nuno Raimundo, Tim Maughan, Marta Popović, Ira Milošević, Kristijan Ramadan.

  DNA repair and autophagy are distinct biological processes vital for cell survival. Although autophagy helps maintain genome stability, there is no evidence of its direct role in the repair of DNA lesions. We discovered that lysosomes process topoisomerase 1 cleavage complexes (TOP1cc) DNA lesions in vertebrates. Selective degradation of TOP1cc by autophagy directs DNA damage repair and cell survival at clinically relevant doses of topoisomerase 1 inhibitors. TOP1cc are exported from the nucleus to lysosomes through a transient alteration of the nuclear envelope and independent of the proteasome. Mechanistically, the autophagy receptor TEX264 acts as a TOP1cc sensor at DNA replication forks, triggering TOP1cc processing by the p97 ATPase and mediating the delivery of TOP1cc to lysosomes in an MRE11-nuclease- and ATR-kinase-dependent manner. We found an evolutionarily conserved role for selective autophagy in DNA repair that enables cell survival, protects genome stability, and is clinically relevant for colorectal cancer patients.

Keywords:  DNA repair; DNA replication; TEX264; colorectal cancer; genome stability; nucleophagy; protein degradation; selective autophagy; topoisomerase 1 cleavage complex; zebrafish

DOI:  https://doi.org/10.1016/j.cell.2024.08.020
EMBO Rep. 2024 Sep 11.

CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction.

Chenliang Zhang, Huanji Xu, Qiulin Tang, Yichun Duan, Hongwei Xia, Huixi Huang, Di Ye, Feng Bi.

  Protein quality control serves as the primary defense mechanism for cells against proteotoxicity induced by proteasome dysfunction. While cells can limit the build-up of ubiquitinated misfolded proteins during proteasome inhibition, the precise mechanism is unclear. Here, we find that protein kinase Ca2+/Calmodulin (CaM)-dependent protein kinase II (CaMKII) maintains proteostasis during proteasome inhibition. We show that proteasome inhibition activates CaMKII, which phosphorylates B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) at residues S173, S377, and S386. Phosphorylated BAG3 activates the heme-regulated inhibitor (HRI)- eukaryotic initiation factor-2α (eIF2α) signaling pathway, suppressing protein synthesis and the production of aggregated ubiquitinated misfolded proteins, ultimately mitigating the proteotoxic crisis. Inhibition of CaMKII exacerbates the accumulation of aggregated misfolded proteins and paraptosis induced by proteasome inhibitors. Based on these findings, we validate that combined targeting of proteasome and CaMKII accelerates tumor cell death and enhances the efficacy of proteasome inhibitors in tumor treatment. Our data unveil a new proteasomal inhibition-induced misfolded protein quality control mechanism and propose a novel therapeutic intervention for proteasome inhibitor-mediated tumor treatment.

Keywords:  BAG3; CaMKII; HRI; Paraptosis; Proteasome Inhibition

DOI:  https://doi.org/10.1038/s44319-024-00248-w
Cell Genom. 2024 Sep 04. pii: S2666-979X(24)00262-3. [Epub ahead of print] 100651

Pooled endogenous protein tagging and recruitment for systematic profiling of protein function.

Yevgeniy V Serebrenik, Deepak Mani, Timothé Maujean, George M Burslem, Ophir Shalem.

  The emerging field of induced proximity therapeutics, which involves designing molecules to bring together an effector and target protein-typically to induce target degradation-is rapidly advancing. However, its progress is constrained by the lack of scalable and unbiased tools to explore effector-target protein interactions. We combine pooled endogenous gene tagging using a ligand-binding domain with generic small-molecule-based recruitment to screen for induction of protein proximity. We apply this methodology to identify effectors for degradation in two orthogonal screens: using fluorescence to monitor target levels and a cellular growth that depends on the degradation of an essential protein. Our screens revealed new effector proteins for degradation, including previously established examples, and converged on members of the C-terminal-to-LisH (CTLH) complex. We introduce a platform for pooled induction of endogenous protein-protein interactions to expand our toolset of effector proteins for protein degradation and other forms of induced proximity.

Keywords:  CTLH; WDR26; functional proteomics; induced proximity; pooled tagging; targeted protein degradation

DOI:  https://doi.org/10.1016/j.xgen.2024.100651
Nat Commun. 2024 Sep 10. 15(1): 7931

RING1 missense variants reveal sensitivity of DNA damage repair to H2A monoubiquitination dosage during neurogenesis.

C W Ryan, S L Regan, E F Mills, B T McGrath, E Gong, Y T Lai, J B Sheingold, K Patel, T Horowitz, A Moccia, Y C Tsan, A Srivastava, S L Bielas.

Polycomb repressive complex 1 (PRC1) modifies chromatin through catalysis of histone H2A lysine 119 monoubiquitination (H2AK119ub1). RING1 and RNF2 interchangeably serve as the catalytic subunit within PRC1. Pathogenic missense variants in PRC1 core components reveal functions of these proteins that are obscured in knockout models. While Ring1a knockout models remain healthy, the microcephaly and neuropsychiatric phenotypes associated with a pathogenic RING1 missense variant implicate unappreciated functions. Using an in vitro model of neurodevelopment, we observe that RING1 contributes to the broad placement of H2AK119ub1, and that its targets overlap with those of RNF2. PRC1 complexes harboring hypomorphic RING1 bind target loci but do not catalyze H2AK119ub1, reducing H2AK119ub1 by preventing catalytically active complexes from accessing the locus. This results in delayed DNA damage repair and cell cycle progression in neural progenitor cells (NPCs). Conversely, reduced H2AK119ub1 due to hypomorphic RING1 does not generate differential expression that impacts NPC differentiation. In contrast, hypomorphic RNF2 generates a greater reduction in H2AK119ub1 that results in both delayed DNA repair and widespread transcriptional changes. These findings suggest that the DNA damage response is more sensitive to H2AK119ub1 dosage change than is regulation of gene expression.

DOI: https://doi.org/10.1038/s41467-024-52292-8
Elife. 2024 Sep 10. pii: RP91002. [Epub ahead of print]12

DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment.

Ran-Der Hwang, YuNing Lu, Qing Tang, Goran Periz, Giho Park, Xiangning Li, Qiwang Xiang, Yang Liu, Tao Zhang, Jiou Wang.

  Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

Keywords:  ES cell; cell culture; fruit flies; human; neuroscience

DOI:  https://doi.org/10.7554/eLife.91002
Elife. 2024 Sep 09. pii: RP99172. [Epub ahead of print]13

Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators.

Roberto Notario Manzano, Thibault Chaze, Eric Rubinstein, Esthel Penard, Mariette Matondo, Chiara Zurzolo, Christel Brou.

  Tunneling nanotubes (TNTs) are open actin- and membrane-based channels, connecting remote cells and allowing direct transfer of cellular material (e.g. vesicles, mRNAs, protein aggregates) from the cytoplasm to the cytoplasm. Although they are important especially, in pathological conditions (e.g. cancers, neurodegenerative diseases), their precise composition and their regulation were still poorly described. Here, using a biochemical approach allowing to separate TNTs from cell bodies and from extracellular vesicles and particles (EVPs), we obtained the full composition of TNTs compared to EVPs. We then focused on two major components of our proteomic data, the CD9 and CD81 tetraspanins, and further investigated their specific roles in TNT formation and function. We show that these two tetraspanins have distinct non-redundant functions: CD9 participates in stabilizing TNTs, whereas CD81 expression is required to allow the functional transfer of vesicles in the newly formed TNTs, possibly by regulating docking to or fusion with the opposing cell.

Keywords:  cell biology; extracellular vesicles; human; tetraspanins; tunneling nanotubes

DOI:  https://doi.org/10.7554/eLife.99172
iScience. 2024 Sep 20. 27(9): 110683

Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis.

Amelie Raby, Sonia Missiroli, Peggy Sanatine, Dominique Langui, Julien Pansiot, Nissai Beaude, Lucie Vezzana, Rachelle Saleh, Martina Marinello, Mireille Laforge, Paolo Pinton, Ana Buj-Bello, Andrea Burgo.

  Mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) emerged to play critical roles in numerous cellular processes, and their dysregulation has been associated to neurodegenerative disorders. Mutations in the SPG4 gene coding for spastin are among the main causes of hereditary spastic paraplegia (HSP). Spastin binds and severs microtubules, and the long isoform of this protein, namely M1, spans the outer leaflet of ER membrane where it interacts with other ER-HSP proteins. Here, we showed that overexpressed M1 spastin localizes in ER-mitochondria intersections and that endogenous spastin accumulates in MERCs. We demonstrated in different cellular models that downregulation of spastin enhances the number of MERCs, alters mitochondrial morphology, and impairs ER and mitochondrial calcium homeostasis. These effects are associated with reduced mitochondrial membrane potential, oxygen species levels, and oxidative metabolism. These findings extend our knowledge on the role of spastin in the ER and suggest MERCs deregulation as potential causes of SPG4-HSP disease.

Keywords:  Biological sciences; Molecular biology; Molecular interaction

DOI:  https://doi.org/10.1016/j.isci.2024.110683
Nat Commun. 2024 Sep 12. 15(1): 7972

Phosphorylation of tau at a single residue inhibits binding to the E3 ubiquitin ligase, CHIP.

Cory M Nadel, Saugat Pokhrel, Kristin Wucherer, Abby Oehler, Aye C Thwin, Koli Basu, Matthew D Callahan, Daniel R Southworth, Daniel A Mordes, Charles S Craik, Jason E Gestwicki.

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimer's disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to tau's accumulation or what mechanisms might be involved. To explore these questions, we focus on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to weaken its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 reveals the mechanism of this clash, allowing design of a mutation (CHIPD134A) that partially restores binding and turnover of pS416 tauC3. We confirm that, in our models, pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a molecular mechanism for how phosphorylation at a discrete site contributes to accumulation of a tau proteoform.

DOI: https://doi.org/10.1038/s41467-024-52075-1
Dev Cell. 2024 Sep 09. pii: S1534-5807(24)00488-X. [Epub ahead of print]59(17): 2273-2274

HOPping to the vacuole: Autophagosome and late endosomes combine to control plant autophagosome degradation.

Jonathan Dragwidge, Amélie Bernard.

In this issue of Developmental Cell, Jiang et al. report that the Arabidopsis HOPS tethering complex subunit VPS41 acts to catalyze the formation of a degradation pathway composed of a hybrid of autophagosomes and late endosomes.

DOI: https://doi.org/10.1016/j.devcel.2024.08.005
Mol Cell. 2024 Sep 05. pii: S1097-2765(24)00700-7. [Epub ahead of print]

RAPIDASH: Tag-free enrichment of ribosome-associated proteins reveals composition dynamics in embryonic tissue, cancer cells, and macrophages.

Teodorus Theo Susanto, Victoria Hung, Andrew G Levine, Yuxiang Chen, Craig H Kerr, Yongjin Yoo, Juan A Oses-Prieto, Lisa Fromm, Zijian Zhang, Travis C Lantz, Kotaro Fujii, Marius Wernig, Alma L Burlingame, Davide Ruggero, Maria Barna.

  Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including Dhx30 and Llph, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed that RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts.

Keywords:  cancer; embryonic development; macrophages; proteomics; ribosome; ribosome heterogeneity; ribosome-associated proteins; translational control

DOI:  https://doi.org/10.1016/j.molcel.2024.08.023
JCI Insight. 2024 Sep 05. pii: e181877. [Epub ahead of print]

Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease.

Amit Prabhakar, Meetu Wadhwa, Rahul Kumar, Prajakta Ghatpande, Aneta Gandjeva, Rubin M Tuder, Brian B Graham, Giorgio Lagna, Akiko Hata.

  Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2 to 3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over one year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR-ISR axis could be a potential therapeutic strategy for PVOD.

Keywords:  Cardiovascular disease; Cell stress; Hypertension; Therapeutics; Vascular biology

DOI:  https://doi.org/10.1172/jci.insight.181877
Life Sci Alliance. 2024 Nov;pii: e202402865. [Epub ahead of print]7(11):

Structural roles of Ump1 and β-subunit propeptides in proteasome biogenesis.

Eric Mark, Paula C Ramos, Fleur Kayser, Jörg Höckendorff, R Jürgen Dohmen, Petra Wendler.

The yeast pre1-1(β4-S142F) mutant accumulates late 20S proteasome core particle precursor complexes (late-PCs). We report a 2.1 Å cryo-EM structure of this intermediate with full-length Ump1 trapped inside, and Pba1-Pba2 attached to the α-ring surfaces. The structure discloses intimate interactions of Ump1 with β2- and β5-propeptides, which together fill most of the antechambers between the α- and β-rings. The β5-propeptide is unprocessed and separates Ump1 from β6 and β7. The β2-propeptide is disconnected from the subunit by autocatalytic processing and localizes between Ump1 and β3. A comparison of different proteasome maturation states reveals that maturation goes along with global conformational changes in the rings, initiated by structuring of the proteolytic sites and their autocatalytic activation. In the pre1-1 strain, β2 is activated first enabling processing of β1-, β6-, and β7-propeptides. Subsequent maturation of β5 and β1 precedes degradation of Ump1, tightening of the complex, and finally release of Pba1-Pba2.

DOI: https://doi.org/10.26508/lsa.202402865
Cell Death Differ. 2024 Sep 12.

Inhibition of nucleo-cytoplasmic proteasome translocation by the aromatic amino acids or silencing Sestrin3-their sensing mediator-is tumor suppressive.

Ido Livneh, Bertrand Fabre, Gilad Goldhirsh, Chen Lulu, Adar Zinger, Yael Shammai Vainer, Maya Kaduri, Aviva Dahan, Tamar Ziv, Avi Schroeder, Yinon Ben-Neriah, Yaniv Zohar, Victoria Cohen-Kaplan, Aaron Ciechanover.

The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response is mediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further show that the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found - using transcriptomic and proteomic analyses - that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3-a mediator of YWF sensing upstream of mTOR-is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular "satiety center" carries therefore potential therapeutic implications for cancer.

DOI: https://doi.org/10.1038/s41418-024-01370-x
Elife. 2024 Sep 11. pii: RP90854. [Epub ahead of print]12

Cannabinoid combination targets NOTCH1-mutated T-cell acute lymphoblastic leukemia through the integrated stress response pathway.

Elazar Besser, Anat Gelfand, Shiri Procaccia, Paula Berman, David Meiri.

  In T-cell acute lymphoblastic leukemia (T-ALL), more than 50% of cases display autoactivation of Notch1 signaling, leading to oncogenic transformation. We have previously identified a specific chemovar of Cannabis that induces apoptosis by preventing Notch1 maturation in leukemia cells. Here, we isolated three cannabinoids from this chemovar that synergistically mimic the effects of the whole extract. Two were previously known, cannabidiol (CBD) and cannabidivarin (CBDV), whereas the third cannabinoid, which we termed 331-18A, was identified and fully characterized in this study. We demonstrated that these cannabinoids act through cannabinoid receptor type 2 and TRPV1 to activate the integrated stress response pathway by depleting intracellular Ca2+. This is followed by increased mRNA and protein expression of ATF4, CHOP, and CHAC1, which is hindered by inhibiting the upstream initiation factor eIF2α. The increased abundance of CHAC1 prevents Notch1 maturation, thereby reducing the levels of the active Notch1 intracellular domain, and consequently decreasing cell viability and increasing apoptosis. Treatment with the three isolated molecules resulted in reduced tumor size and weight in vivo and slowed leukemia progression in mice models. Altogether, this study elucidated the mechanism of action of three distinct cannabinoids in modulating the Notch1 pathway, and constitutes an important step in the establishment of a new therapy for treating NOTCH1-mutated diseases and cancers such as T-ALL.

Keywords:  ISR; Notch1; T-ALL; cancer biology; cannabinoids; integrated stress response; leukemia; mouse

DOI:  https://doi.org/10.7554/eLife.90854
Sci Adv. 2024 Sep 13. 10(37): eadp4119

Antibiotics damage the colonic mucus barrier in a microbiota-independent manner.

Jasmin Sawaed, Lilach Zelik, Yehonatan Levin, Rachel Feeney, Maria Naama, Ateret Gordon, Mor Zigdon, Elad Rubin, Shahar Telpaz, Sonia Modilevsky, Shira Ben-Simon, Aya Awad, Sarina Harshuk-Shabso, Meital Nuriel-Ohayon, Michal Werbner, Bjoern O Schroeder, Amir Erez, Shai Bel.

Antibiotic use is a risk factor for development of inflammatory bowel diseases (IBDs). IBDs are characterized by a damaged mucus layer, which does not separate the intestinal epithelium from the microbiota. Here, we hypothesized that antibiotics affect the integrity of the mucus barrier, which allows bacterial penetrance and predisposes to intestinal inflammation. We found that antibiotic treatment led to breakdown of the colonic mucus barrier and penetration of bacteria into the mucus layer. Using fecal microbiota transplant, RNA sequencing followed by machine learning, ex vivo mucus secretion measurements, and antibiotic treatment of germ-free mice, we determined that antibiotics induce endoplasmic reticulum stress in the colon that inhibits colonic mucus secretion in a microbiota-independent manner. This antibiotic-induced mucus secretion flaw led to penetration of bacteria into the colonic mucus layer, translocation of microbial antigens into circulation, and exacerbation of ulcerations in a mouse model of IBD. Thus, antibiotic use might predispose to intestinal inflammation by impeding mucus production.

DOI: https://doi.org/10.1126/sciadv.adp4119
Mol Cell. 2024 Sep 05. pii: S1097-2765(24)00665-8. [Epub ahead of print]84(17): 3169-3171

Proteasomes safeguard the plant stress granule homeostasis.

Qi Chen, Xiaoxin Chen, Peiguo Yang.

In this issue of Molecular Cell, Xie et al.1 revealed that the proteasome is a constitutive component of plant stress granules (SGs), and that enhanced proteolytic activity is essential for efficient SG disassembly and plant survival during the stress response.

DOI: https://doi.org/10.1016/j.molcel.2024.08.011
Nat Commun. 2024 Sep 11. 15(1): 7946

Automated design of multi-target ligands by generative deep learning.

Laura Isigkeit, Tim Hörmann, Espen Schallmayer, Katharina Scholz, Felix F Lillich, Johanna H M Ehrler, Benedikt Hufnagel, Jasmin Büchner, Julian A Marschner, Jörg Pabel, Ewgenij Proschak, Daniel Merk.

Generative deep learning models enable data-driven de novo design of molecules with tailored features. Chemical language models (CLM) trained on string representations of molecules such as SMILES have been successfully employed to design new chemical entities with experimentally confirmed activity on intended targets. Here, we probe the application of CLM to generate multi-target ligands for designed polypharmacology. We capitalize on the ability of CLM to learn from small fine-tuning sets of molecules and successfully bias the model towards designing drug-like molecules with similarity to known ligands of target pairs of interest. Designs obtained from CLM after pooled fine-tuning are predicted active on both proteins of interest and comprise pharmacophore elements of ligands for both targets in one molecule. Synthesis and testing of twelve computationally favored CLM designs for six target pairs reveals modulation of at least one intended protein by all selected designs with up to double-digit nanomolar potency and confirms seven compounds as designed dual ligands. These results corroborate CLM for multi-target de novo design as source of innovation in drug discovery.

DOI: https://doi.org/10.1038/s41467-024-52060-8
EMBO J. 2024 Sep 10.

The fitness cost of spurious phosphorylation.

David Bradley, Alexander Hogrebe, Rohan Dandage, Alexandre K Dubé, Mario Leutert, Ugo Dionne, Alexis Chang, Judit Villén, Christian R Landry.

  The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known. Here, we use Saccharomyces cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, the resulting tyrosine phosphorylation is biologically spurious. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3500 proteins. The number of spurious pY sites generated correlates strongly with decreased growth, and we predict over 1000 pY events to be deleterious. However, we also find that many of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with tyrosine kinases. Our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

Keywords:  Evolution; Gene Editing; Phosphoproteomics; Protein Kinases; Structural Bioinformatics

DOI:  https://doi.org/10.1038/s44318-024-00200-7