bims-proteo 2024-01-07 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024‒01‒07
37 papers selected by
Eric Chevet, INSERM

The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells.
UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation.
Co-opting the E3 ligase KLHDC2 for targeted protein degradation by small molecules.
The ER membrane protein complex restricts mitophagy by controlling BNIP3 turnover.
The ubiquitin-conjugating enzyme UBE2D/eff maintains a youthful proteome and ensures protein quality control during aging.
Nuclear Hsp104 safeguards the dormant translation machinery during quiescence.
Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2.
USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting ubiquitin signals from endosomes.
DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance.
UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4.
The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center.
Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad.
IGHMBP2 deletion suppresses translation and activates the integrated stress response.
TMEM55B links autophagy flux, lysosomal repair, and TFE3 activation in response to oxidative stress.
CAND1 inhibits Cullin-2-RING ubiquitin ligases for enhanced substrate specificity.
B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.
Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells.
Fission-independent compartmentalization of mitochondria during budding yeast cell division.
Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection.
Bivalent inhibitors of the BTB E3 ligase KEAP1 enable instant NRF2 activation to suppress acute inflammatory response.
The STX17-SNAP47-VAMP7/VAMP8 complex is the default SNARE complex mediating autophagosome-lysosome fusion.
Proximity labeling reveals dynamic changes in the SQSTM1 protein network.
Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.
4R MAPT/tau drives endolysosomal and autophagy dysfunction in frontotemporal dementia.
Recruitment of trimeric eIF2 by phosphatase non-catalytic subunit PPP1R15B.
eIF4E1b is a non-canonical eIF4E protecting maternal dormant mRNAs.
Induced protein degradation for therapeutics: past, present, and future.
The ubiquitin ligases Cbl and Cbl-b regulate macrophage growth by controlling CSF-1R import into macropinosomes.
Derailed protein turnover in the aging mammalian brain.
A beneficial adaptive role for CHOP in driving cell fate selection during ER stress.
Experimental determination and mathematical modeling of standard shapes of forming autophagosomes.
Self-Sorting Collagen Heterotrimers.
A proteolytic AAA+ machine poised to unfold a protein substrate.
TREX2 deficiency suppresses spontaneous and genotoxin-associated mutagenesis.
Degraders Upgraded: The Rise of PROTACs in Haematological Malignancies.
Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.
Variable PD-1 glycosylation modulates the activity of immune checkpoint inhibitors.

EMBO J. 2023 Dec 18.

The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells.

Lisa Neidhardt, Eva Cloots, Natalie Friemel, Caroline A M Weiss, Heather P Harding, Stephen H McLaughlin, Sophie Janssens, David Ron.

  Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1β) with the luminal domain of IRE1β deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1β/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1β/α chimera. In vitro, AGR2 actively de-stabilised the IRE1β luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1β-AGR2 couple suggest that active repression of IRE1β by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.

Keywords:  Endoplasmic Reticulum (ER); Molecular Chaperones; Mucin; Protein Multimerisation; Unfolded Protein Response (UPR)

DOI:  https://doi.org/10.1038/s44318-023-00014-z
EMBO Rep. 2024 Jan 02.

UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation.

Brittany A Ahlstedt, Rakesh Ganji, Sirisha Mukkavalli, Joao A Paulo, Steve P Gygi, Malavika Raman.

  ER protein homeostasis (proteostasis) is essential for proper folding and maturation of proteins in the secretory pathway. Loss of ER proteostasis can lead to the accumulation of misfolded or aberrant proteins in the ER and triggers the unfolded protein response (UPR). In this study, we find that the p97 adaptor UBXN1 is an important negative regulator of the UPR. Loss of UBXN1 sensitizes cells to ER stress and activates the UPR. This leads to widespread upregulation of the ER stress transcriptional program. Using comparative, quantitative proteomics we show that deletion of UBXN1 results in a significant enrichment of proteins involved in ER-quality control processes including those involved in protein folding and import. Notably, we find that loss of UBXN1 does not perturb p97-dependent ER-associated degradation (ERAD). Our studies indicate that loss of UBXN1 increases translation in both resting and ER-stressed cells. Surprisingly, this process is independent of p97 function. Taken together, our studies have identified a new role for UBXN1 in repressing translation and maintaining ER proteostasis in a p97 independent manner.

Keywords:  ER Stress; Translation; Ubiquitin; Unfolded Protein Response; p97

DOI:  https://doi.org/10.1038/s44319-023-00027-z
Nat Struct Mol Biol. 2024 Jan 04.

Co-opting the E3 ligase KLHDC2 for targeted protein degradation by small molecules.

Christopher M Hickey, Katherine M Digianantonio, Kurt Zimmermann, Alicia Harbin, Connor Quinn, Avani Patel, Peter Gareiss, Amanda Chapman, Bernadette Tiberi, Jennifer Dobrodziej, John Corradi, Angela M Cacace, David R Langley, Miklós Békés.

Targeted protein degradation (TPD) by PROTAC (proteolysis-targeting chimera) and molecular glue small molecules is an emerging therapeutic strategy. To expand the roster of E3 ligases that can be utilized for TPD, we describe the discovery and biochemical characterization of small-molecule ligands targeting the E3 ligase KLHDC2. Furthermore, we functionalize these KLHDC2-targeting ligands into KLHDC2-based BET-family and AR PROTAC degraders and demonstrate KLHDC2-dependent target-protein degradation. Additionally, we offer insight into the assembly of the KLHDC2 E3 ligase complex. Using biochemical binding studies, X-ray crystallography and cryo-EM, we show that the KLHDC2 E3 ligase assembles into a dynamic tetramer held together via its own C terminus, and that this assembly can be modulated by substrate and ligand engagement.

DOI: https://doi.org/10.1038/s41594-023-01146-w
EMBO J. 2024 Jan;43(1): 32-60

The ER membrane protein complex restricts mitophagy by controlling BNIP3 turnover.

Jose M Delgado, Logan Wallace Shepard, Sarah W Lamson, Samantha L Liu, Christopher J Shoemaker.

  Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. This screen revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system and the ubiquitin-proteosome system regulated BNIP3 independently. Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. More broadly, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that tightly regulate endogenous tail-anchored protein localization.

Keywords:  BNIP3; EMC; Mitophagy; Secretory Pathway; TA Protein

DOI:  https://doi.org/10.1038/s44318-023-00006-z
bioRxiv. 2023 Dec 13. pii: 2023.12.12.571303. [Epub ahead of print]

The ubiquitin-conjugating enzyme UBE2D/eff maintains a youthful proteome and ensures protein quality control during aging.

Liam C Hunt, Kudzai Nyamkondiwa, Anna Stephan, Jianqin Jiao, Kanisha Kavdia, Vishwajeeth Pagala, Junmin Peng, Fabio Demontis.

Ubiquitin-conjugating enzymes (E2s) are key for regulating protein function and turnover via ubiquitination but it remains undetermined which E2s maintain proteostasis during aging. Here, we find that E2s have diverse roles in handling a model aggregation-prone protein (huntingtin-polyQ) in the Drosophila retina: while some E2s mediate aggregate assembly, UBE2D/effete (eff) and other E2s are required for huntingtin-polyQ degradation. UBE2D/eff is key for proteostasis also in skeletal muscle: eff protein levels decline with aging, and muscle-specific eff knockdown causes an accelerated buildup in insoluble poly-ubiquitinated proteins (which progressively accumulate with aging) and shortens lifespan. Transgenic expression of human UBE2D2, homologous to eff, partially rescues the lifespan and proteostasis deficits caused by muscle-specific eff RNAi by re-establishing the physiological levels of eff RNAi -regulated proteins. Interestingly, UBE2D/eff knockdown in young age reproduces many of the proteomic changes that normally occur in old muscles, suggesting that the decrease in UBE2D/eff protein levels that occurs with aging contributes to reshaping the composition of the muscle proteome. Altogether, these findings indicate that UBE2D/eff is a key E2 ubiquitin-conjugating enzyme for maintaining a youthful proteome and for ensuring protein quality control during aging.

DOI: https://doi.org/10.1101/2023.12.12.571303
Nat Commun. 2024 Jan 05. 15(1): 315

Nuclear Hsp104 safeguards the dormant translation machinery during quiescence.

Verena Kohler, Andreas Kohler, Lisa Larsson Berglund, Xinxin Hao, Sarah Gersing, Axel Imhof, Thomas Nyström, Johanna L Höög, Martin Ott, Claes Andréasson, Sabrina Büttner.

The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.

DOI: https://doi.org/10.1038/s41467-023-44538-8
EMBO J. 2023 Dec 20.

Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2.

Eva Cloots, Phaedra Guilbert, Mathias Provost, Lisa Neidhardt, Evelien Van de Velde, Farzaneh Fayazpour, Delphine De Sutter, Savvas N Savvides, Sven Eyckerman, Sophie Janssens.

  Intestinal goblet cells are secretory cells specialized in the production of mucins, and as such are challenged by the need for efficient protein folding. Goblet cells express Inositol-Requiring Enzyme-1β (IRE1β), a unique sensor in the unfolded protein response (UPR), which is part of an adaptive mechanism that regulates the demands of mucin production and secretion. However, how IRE1β activity is tuned to mucus folding load remains unknown. We identified the disulfide isomerase and mucin chaperone AGR2 as a goblet cell-specific protein that crucially regulates IRE1β-, but not IRE1α-mediated signaling. AGR2 binding to IRE1β disrupts IRE1β oligomerization, thereby blocking its downstream endonuclease activity. Depletion of endogenous AGR2 from goblet cells induces spontaneous IRE1β activation, suggesting that alterations in AGR2 availability in the endoplasmic reticulum set the threshold for IRE1β activation. We found that AGR2 mutants lacking their catalytic cysteine, or displaying the disease-associated mutation H117Y, were no longer able to dampen IRE1β activity. Collectively, these results demonstrate that AGR2 is a central chaperone regulating the goblet cell UPR by acting as a rheostat of IRE1β endonuclease activity.

Keywords:  AGR2; Goblet Cells; IRE1β; Mucus Homeostasis; Unfolded Protein Response (UPR)

DOI:  https://doi.org/10.1038/s44318-023-00015-y
J Cell Biol. 2024 Mar 04. pii: e202306013. [Epub ahead of print]223(3):

USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting ubiquitin signals from endosomes.

Akinori Endo, Toshiaki Fukushima, Chikage Takahashi, Hikaru Tsuchiya, Fumiaki Ohtake, Sayaka Ono, Tony Ly, Yukiko Yoshida, Keiji Tanaka, Yasushi Saeki, Masayuki Komada.

K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1-NF-κB and Keap1-Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes.

DOI: https://doi.org/10.1083/jcb.202306013
Nat Commun. 2024 Jan 04. 15(1): 275

DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance.

Martin Schröder, Martin Renatus, Xiaoyou Liang, Fabian Meili, Thomas Zoller, Sandrine Ferrand, Francois Gauter, Xiaoyan Li, Frederic Sigoillot, Scott Gleim, Therese-Marie Stachyra, Jason R Thomas, Damien Begue, Maryam Khoshouei, Peggy Lefeuvre, Rita Andraos-Rey, BoYee Chung, Renate Ma, Benika Pinch, Andreas Hofmann, Markus Schirle, Niko Schmiedeberg, Patricia Imbach, Delphine Gorses, Keith Calkins, Beatrice Bauer-Probst, Magdalena Maschlej, Matt Niederst, Rob Maher, Martin Henault, John Alford, Erik Ahrne, Luca Tordella, Greg Hollingworth, Nicolas H Thomä, Anna Vulpetti, Thomas Radimerski, Philipp Holzer, Seth Carbonneau, Claudio R Thoma.

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.

DOI: https://doi.org/10.1038/s41467-023-44237-4
Nat Struct Mol Biol. 2024 Jan 05.

UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4.

Lucy Barnsby-Greer, Peter D Mabbitt, Marc-Andre Dery, Daniel R Squair, Nicola T Wood, Frederic Lamoliatte, Sven M Lange, Satpal Virdee.

UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a 'hemiRING' zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2-E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2∼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.

DOI: https://doi.org/10.1038/s41594-023-01192-4
Nat Chem Biol. 2024 Jan 03.

The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center.

João P L Coelho, Matthew C J Yip, Keely Oltion, Jack Taunton, Sichen Shao.

Translation termination is an essential cellular process, which is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins from ribosomes and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. However, the mechanism of action of SRI-41315 on eRF1 and translation is not known. Here we report cryo-EM structures showing that SRI-41315 acts as a metal-dependent molecular glue between the N domain of eRF1 responsible for stop codon recognition and the ribosomal subunit interface near the decoding center. Retention of eRF1 on ribosomes by SRI-41315 leads to ribosome collisions, eRF1 ubiquitylation and a higher frequency of translation termination at near-cognate stop codons. Our findings reveal a new mechanism of release factor inhibition and additional implications for pharmacologically targeting eRF1.

DOI: https://doi.org/10.1038/s41589-023-01521-0
Nat Commun. 2024 Jan 02. 15(1): 134

Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad.

Hyunju Cho, Yumeng Liu, SangYoon Chung, Sowmya Chandrasekar, Shimon Weiss, Shu-Ou Shan.

Membrane protein biogenesis poses acute challenges to protein homeostasis, and how they are selectively escorted to the target membrane is not well understood. Here we address this question in the guided-entry-of-tail-anchored protein (GET) pathway, in which tail-anchored membrane proteins (TAs) are relayed through an Hsp70-Sgt2-Get3 chaperone triad for targeting to the endoplasmic reticulum. We show that the Hsp70 ATPase cycle and TA substrate drive dimeric Sgt2 from a wide-open conformation to a closed state, in which TAs are protected by both substrate binding domains of Sgt2. Get3 is privileged to receive TA from closed Sgt2, whereas off-pathway chaperones remove TAs from open Sgt2. Sgt2 closing is less favorable with suboptimal GET substrates, which are rejected during or after the Hsp70-to-Sgt2 handover. Our results demonstrate how fine-tuned conformational dynamics in Sgt2 enable hydrophobic TAs to be effectively funneled onto their dedicated targeting factor while also providing a mechanism for substrate selection.

DOI: https://doi.org/10.1038/s41467-023-44260-5
bioRxiv. 2023 Dec 12. pii: 2023.12.11.571166. [Epub ahead of print]

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

Jesslyn E Park, Hetvee Desai, José Liboy-Lugo, Sohyun Gu, Ziad Jowhar, Albert Xu, Stephen N Floor.

IGHMBP2 is a non-essential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 upregulation. With recent studies showing the ISR can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

DOI: https://doi.org/10.1101/2023.12.11.571166
Nat Commun. 2024 Jan 02. 15(1): 93

TMEM55B links autophagy flux, lysosomal repair, and TFE3 activation in response to oxidative stress.

Eutteum Jeong, Rose Willett, Alberto Rissone, Martina La Spina, Rosa Puertollano.

Lysosomes have emerged as critical regulators of cellular homeostasis. Here we show that the lysosomal protein TMEM55B contributes to restore cellular homeostasis in response to oxidative stress by three different mechanisms: (1) TMEM55B mediates NEDD4-dependent PLEKHM1 ubiquitination, causing PLEKHM1 proteasomal degradation and halting autophagosome/lysosome fusion; (2) TMEM55B promotes recruitment of components of the ESCRT machinery to lysosomal membranes to stimulate lysosomal repair; and (3) TMEM55B sequesters the FLCN/FNIP complex to facilitate translocation of the transcription factor TFE3 to the nucleus, allowing expression of transcriptional programs that enable cellular adaptation to stress. Knockout of tmem55 genes in zebrafish embryos increases their susceptibility to oxidative stress, causing early death of tmem55-KO animals in response to arsenite toxicity. Altogether, our work identifies a role for TMEM55B as a molecular sensor that coordinates autophagosome degradation, lysosomal repair, and activation of stress responses.

DOI: https://doi.org/10.1038/s41467-023-44316-6
Nat Struct Mol Biol. 2024 Jan 04.

CAND1 inhibits Cullin-2-RING ubiquitin ligases for enhanced substrate specificity.

Kankan Wang, Stephanie Diaz, Lihong Li, Jeremy R Lohman, Xing Liu.

Through targeting essential cellular regulators for ubiquitination and serving as a major platform for discovering proteolysis-targeting chimera (PROTAC) drugs, Cullin-2 (CUL2)-RING ubiquitin ligases (CRL2s) comprise an important family of CRLs. The founding members of CRLs, the CUL1-based CRL1s, are known to be activated by CAND1, which exchanges the variable substrate receptors associated with the common CUL1 core and promotes the dynamic assembly of CRL1s. Here we find that CAND1 inhibits CRL2-mediated protein degradation in human cells. This effect arises due to altered binding kinetics, involving CAND1 and CRL2VHL, as we illustrate that CAND1 dramatically increases the dissociation rate of CRL2s but barely accelerates the assembly of stable CRL2s. Using PROTACs that differently recruit neo-substrates to CRL2VHL, we demonstrate that the inhibitory effect of CAND1 helps distinguish target proteins with different affinities for CRL2s, presenting a mechanism for selective protein degradation with proper pacing in the changing cellular environment.

DOI: https://doi.org/10.1038/s41594-023-01167-5
EMBO J. 2023 Dec 14.

B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Esther N Park, Timur Mackens-Kiani, Rebekah Berhane, Hanna Esser, Chimeg Erdenebat, A Maxwell Burroughs, Otto Berninghausen, L Aravind, Roland Beckmann, Rachel Green, Allen R Buskirk.

  Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.

Keywords:  ABC ATPase; B. subtilis; MutS2; Ribosome Collisions; Ribosome Quality Control (RQC)

DOI:  https://doi.org/10.1038/s44318-023-00010-3
Cell Rep. 2023 Dec 28. pii: S2211-1247(23)01627-3. [Epub ahead of print]43(1): 113615

Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells.

Mehdi Amiri, Stephen J Kiniry, Anthony P Possemato, Niaz Mahmood, Tayebeh Basiri, Catherine R Dufour, Negar Tabatabaei, Qiyun Deng, Michael A Bellucci, Keerthana Harwalkar, Matthew P Stokes, Vincent Giguère, Randal J Kaufman, Yojiro Yamanaka, Pavel V Baranov, Soroush Tahmasebi, Nahum Sonenberg.

  The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/β) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.

Keywords:  Stem cell research; embryonic stem cells; p-eIF2α; pluripotency; ribosome profiling; translational control

DOI:  https://doi.org/10.1016/j.celrep.2023.113615
J Cell Biol. 2024 Mar 04. pii: e202211048. [Epub ahead of print]223(3):

Fission-independent compartmentalization of mitochondria during budding yeast cell division.

Saori R Yoshii, Yves Barral.

Lateral diffusion barriers compartmentalize membranes to generate polarity or asymmetrically partition membrane-associated macromolecules. Budding yeasts assemble such barriers in the endoplasmic reticulum (ER) and the outer nuclear envelope at the bud neck to retain aging factors in the mother cell and generate naïve and rejuvenated daughter cells. However, little is known about whether other organelles are similarly compartmentalized. Here, we show that the membranes of mitochondria are laterally compartmentalized at the bud neck and near the cell poles. The barriers in the inner mitochondrial membrane are constitutive, whereas those in the outer membrane form in response to stresses. The strength of mitochondrial diffusion barriers is regulated positively by spatial cues from the septin axis and negatively by retrograde (RTG) signaling. These data indicate that mitochondria are compartmentalized in a fission-independent manner. We propose that these diffusion barriers promote mitochondrial polarity and contribute to mitochondrial quality control.

DOI: https://doi.org/10.1083/jcb.202211048
iScience. 2023 Dec 15. 26(12): 108570

Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection.

Jessica Vetters, Mary van Helden, Clint De Nolf, Sofie Rennen, Eva Cloots, Evelien Van De Velde, Farzaneh Fayazpour, Justine Van Moorleghem, Manon Vanheerswynghels, Karl Vergote, Louis Boon, Eric Vivier, Bart N Lambrecht, Sophie Janssens.

  The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency of IRE1 and its downstream transcription factor XBP1 in NKp46+ NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1-sufficient Ly49H+ NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that IRE1/XBP1 activation is required during vigorous NK cell proliferation early upon viral infection, as part of a canonical UPR response.

Keywords:  Immunology; Microbiology; Transcriptomics; Virology

DOI:  https://doi.org/10.1016/j.isci.2023.108570
Cell Chem Biol. 2023 Dec 21. pii: S2451-9456(23)00435-X. [Epub ahead of print]

Bivalent inhibitors of the BTB E3 ligase KEAP1 enable instant NRF2 activation to suppress acute inflammatory response.

Mengchen Lu, Jianai Ji, Yifei Lv, Jing Zhao, Yuting Liu, Qiong Jiao, Tian Liu, Yi Mou, Qidong You, Zhengyu Jiang.

  Most BTB-containing E3 ligases homodimerize to recognize a single substrate by engaging multiple degrons, represented by E3 ligase KEAP1 dimer and its substrate NRF2. Inactivating KEAP1 to hinder ubiquitination-dependent NRF2 degradation activates NRF2. While various KEAP1 inhibitors have been reported, all reported inhibitors bind to KEAP1 in a monovalent fashion and activate NRF2 in a lagging manner. Herein, we report a unique bivalent KEAP1 inhibitor, biKEAP1 (3), that engages cellular KEAP1 dimer to directly release sequestered NRF2 protein, leading to an instant NRF2 activation. 3 promotes the nuclear translocation of NRF2, directly suppressing proinflammatory cytokine transcription. Data from in vivo experiments showed that 3, with unprecedented potency, reduced acute inflammatory burden in several acute inflammation models in a timely manner. Our findings demonstrate that the bivalent KEAP1 inhibitor can directly enable sequestered substrate NRF2 to suppress inflammatory transcription response and dampen various acute inflammation injuries.

Keywords:  BTB E3 ligase; KEAP1; NRF2 activation; acute inflammation; bivalent inhibitor

DOI:  https://doi.org/10.1016/j.chembiol.2023.12.005
Cell Res. 2024 Jan 05.

The STX17-SNAP47-VAMP7/VAMP8 complex is the default SNARE complex mediating autophagosome-lysosome fusion.

Fenglei Jian, Shen Wang, Rui Tian, Yufen Wang, Chuangpeng Li, Yan Li, Shixuan Wang, Chao Fang, Cong Ma, Yueguang Rong.

Autophagosome-lysosome fusion mediated by SNARE complexes is an essential step in autophagy. Two SNAP29-containing SNARE complexes have been extensively studied in starvation-induced bulk autophagy, while the relevant SNARE complexes in other types of autophagy occurring under non-starvation conditions have been overlooked. Here, we found that autophagosome-lysosome fusion in selective autophagy under non-starvation conditions does not require SNAP29-containing SNARE complexes, but requires the STX17-SNAP47-VAMP7/VAMP8 SNARE complex. Further, the STX17-SNAP47-VAMP7/VAMP8 SNARE complex also functions in starvation-induced autophagy. SNAP47 is recruited to autophagosomes following concurrent detection of ATG8s and PI(4,5)P2 via its Pleckstrin homology domain. By contrast, SNAP29-containing SNAREs are excluded from selective autophagy due to inactivation by O-GlcNAcylation under non-starvation conditions. These findings depict a previously unknown, default SNARE complex responsible for autophagosome-lysosome fusion in both selective and bulk autophagy, which could guide research and therapeutic development in autophagy-related diseases.

DOI: https://doi.org/10.1038/s41422-023-00916-x
bioRxiv. 2023 Dec 13. pii: 2023.12.12.571324. [Epub ahead of print]

Proximity labeling reveals dynamic changes in the SQSTM1 protein network.

Alejandro N Rondón Ortiz, Lushuang Zhang, Peter E A Ash, Avik Basu, Sambhavi Puri, Sophie J F van der Spek, Luke Dorrian, Andrew Emili, Benjamin Wolozin.

Sequestosome1 (SQSTM1) is an autophagy receptor that mediates degrada4on of intracellular cargo, including protein aggregates, through mul4ple protein interac4ons. These interac4ons form the SQSTM1 protein network that are mediated by SQSTM1 func4onal interac4on domains, which include LIR, PB1, UBA and KIR. Despite various abempts to unravel the complexity of the SQSTM1 protein network, our understanding of the rela4onship of various components in cellular physiology and disease states con4nues to evolve. To inves4gate the SQSTM1 protein interac4on network, we performed proximity profile labeling by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including: produc4on of SQSTM1 intracellular bodies, binding to known SQSTM1 interac4ng partners via defined func4onal SQSTM1 interac4ng domains and capture of novel SQSTM1 interactors. Strikingly, aggregated tau protein altered the protein interac4on network of SQSTM1 to include many stress-associated proteins. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 func4on in cellular physiology and disease state. ProteomeXchange Consor4um PRIDE Submission: Submission details: Project Name: Proximity labeling reveals dynamic changes in the SQSTM1 protein network. Project accession: PXD047725 Project DOI: Not applicable.

DOI: https://doi.org/10.1101/2023.12.12.571324
EMBO J. 2024 Jan 02.

Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.

Fatima Amer-Sarsour, Daniel Falik, Yevgeny Berdichevsky, Alina Kordonsky, Sharbel Eid, Tatiana Rabinski, Hasan Ishtayeh, Stav Cohen-Adiv, Itzhak Braverman, Sergiu C Blumen, Tal Laviv, Gali Prag, Gad D Vatine, Avraham Ashkenazi.

  Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.

Keywords:  Autonomic Nervous System; Congenital Central Hypoventilation Syndrome; Trinucleotide Repeats; Ubiquitin Transfer System; Ubiquitin-Activating Enzyme

DOI:  https://doi.org/10.1038/s44318-023-00018-9
Autophagy. 2024 Jan 04.

4R MAPT/tau drives endolysosomal and autophagy dysfunction in frontotemporal dementia.

Christy Hung, Rickie Patani.

  Dysfunction of the neuronal endolysosome and macroautophagy/autophagy pathway is emerging as an important pathogenic mechanism in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The VCP (valosin-containing protein) gene is of significant relevance, directly implicated in both FTD and ALS. In our recent study, we used patient-derived stem cells to study the effects of VCP mutations on the endolysosome and autophagy system in human cortical excitatory neurons. We found that VCP mutations cause an abnormal accumulation of enlarged endosomes and lysosomes, accompanied by reduced autophagy flux. VCP mutations also lead to the spatial dissociation of intra-nuclear RNA-binding proteins, FUS and SFPQ, which correlates with alternative splicing of the MAPT pre-mRNA and increased MAPT/tau phosphorylation. Importantly, we found that an increase in the 4 R-MAPT/tau isoform is sufficient to drive toxic changes in healthy human cortical excitatory neurons, including MAPT/tau hyperphosphorylation, endolysosomal dysfunction, lysosomal membrane rupture, endoplasmic reticulum stress, and apoptosis. Together, our data suggest that endolysosomal and autophagy dysfunction could represent a convergent pathogenic "design principle" shared by both FTD and ALS.

Keywords:  Autophagy; endosomes; lysosome; tau; tauopathy

DOI:  https://doi.org/10.1080/15548627.2023.2300917
Mol Cell. 2023 Dec 27. pii: S1097-2765(23)01030-4. [Epub ahead of print]

Recruitment of trimeric eIF2 by phosphatase non-catalytic subunit PPP1R15B.

Agnieszka Fatalska, George Hodgson, Stefan M V Freund, Sarah L Maslen, Tomos Morgan, Sigurdur R Thorkelsson, Marjon van Slegtenhorst, Sonja Lorenz, Antonina Andreeva, Laura Donker Kaat, Anne Bertolotti.

  Regulated protein phosphorylation controls most cellular processes. The protein phosphatase PP1 is the catalytic subunit of many holoenzymes that dephosphorylate serine/threonine residues. How these enzymes recruit their substrates is largely unknown. Here, we integrated diverse approaches to elucidate how the PP1 non-catalytic subunit PPP1R15B (R15B) captures its full trimeric eIF2 substrate. We found that the substrate-recruitment module of R15B is largely disordered with three short helical elements, H1, H2, and H3. H1 and H2 form a clamp that grasps the substrate in a region remote from the phosphorylated residue. A homozygous N423D variant, adjacent to H1, reducing substrate binding and dephosphorylation was discovered in a rare syndrome with microcephaly, developmental delay, and intellectual disability. These findings explain how R15B captures its 125 kDa substrate by binding the far end of the complex relative to the phosphosite to present it for dephosphorylation by PP1, a paradigm of broad relevance.

Keywords:  PP1; PPP1R15B; intrinsically disordered proteins; mass spectrometry; missense variants; nuclear magnetic resonance spectroscopy; phosphatase; structural proteomics; syndrome

DOI:  https://doi.org/10.1016/j.molcel.2023.12.011
EMBO Rep. 2023 Dec 14.

eIF4E1b is a non-canonical eIF4E protecting maternal dormant mRNAs.

Laura Lorenzo-Orts, Marcus Strobl, Benjamin Steinmetz, Friederike Leesch, Carina Pribitzer, Josef Roehsner, Michael Schutzbier, Gerhard Dürnberger, Andrea Pauli.

  Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.

Keywords:  Maternal mRNAs; P-bodies; Translational Regulation; Zebrafish; eIF4E1b

DOI:  https://doi.org/10.1038/s44319-023-00006-4
J Clin Invest. 2024 Jan 02. pii: e175265. [Epub ahead of print]134(1):

Induced protein degradation for therapeutics: past, present, and future.

Hojong Yoon, Justine C Rutter, Yen-Der Li, Benjamin L Ebert.

The concept of induced protein degradation by small molecules has emerged as a promising therapeutic strategy that is particularly effective in targeting proteins previously considered "undruggable." Thalidomide analogs, employed in the treatment of multiple myeloma, stand as prime examples. These compounds serve as molecular glues, redirecting the CRBN E3 ubiquitin ligase to degrade myeloma-dependency factors, IKZF1 and IKZF3. The clinical success of thalidomide analogs demonstrates the therapeutic potential of induced protein degradation. Beyond molecular glue degraders, several additional modalities to trigger protein degradation have been developed and are currently under clinical evaluation. These include heterobifunctional degraders, polymerization-induced degradation, ligand-dependent degradation of nuclear hormone receptors, disruption of protein interactions, and various other strategies. In this Review, we will provide a concise overview of various degradation modalities, their clinical applications, and potential future directions in the field of protein degradation.

DOI: https://doi.org/10.1172/JCI175265
Mol Biol Cell. 2024 Jan 03. mbcE23090345

The ubiquitin ligases Cbl and Cbl-b regulate macrophage growth by controlling CSF-1R import into macropinosomes.

Lu Huang, Natalie W Thiex, Jieqiong Lou, Gulzar Ahmad, Wei An, Shalini T Low-Nam, Jason G Kerkviet, Hamid Band, Adam D Hoppe.

Ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKOmacrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNAseq comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.

DOI: https://doi.org/10.1091/mbc.E23-09-0345
Mol Syst Biol. 2024 Jan 05.

Derailed protein turnover in the aging mammalian brain.

Nalini R Rao, Arun Upadhyay, Jeffrey N Savas.

  Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Keywords:  Aging; proteasome; protein turnover; quantitative proteomics; stable isotope labeling

DOI:  https://doi.org/10.1038/s44320-023-00009-2
EMBO Rep. 2024 Jan 02.

A beneficial adaptive role for CHOP in driving cell fate selection during ER stress.

Kaihua Liu, Chaoxian Zhao, Reed C Adajar, Diane DeZwaan-McCabe, D Thomas Rutkowski.

  Cellular stresses elicit signaling cascades that are capable of either mitigating the inciting dysfunction or initiating cell death. During endoplasmic reticulum (ER) stress, the transcription factor CHOP is widely recognized to promote cell death. However, it is not clear whether CHOP also has a beneficial role during adaptation. Here, we combine a new, versatile, genetically modified Chop allele with single cell analysis and with stresses of physiological intensity, to rigorously examine the contribution of CHOP to cell fate. Paradoxically, we find that CHOP promotes death in some cells, but proliferation-and hence recovery-in others. Strikingly, this function of CHOP confers to cells a stress-specific competitive growth advantage. The dynamics of CHOP expression and UPR activation at the single cell level suggest that CHOP maximizes UPR activation, which in turn favors stress resolution, subsequent UPR deactivation, and proliferation. Taken together, these findings suggest that CHOP's function can be better described as a "stress test" that drives cells into either of two mutually exclusive fates-adaptation or death-during stresses of physiological intensity.

Keywords:  Adaptation; Cell Fate; ER Stress; Cell Proliferation; Unfolded Protein Response

DOI:  https://doi.org/10.1038/s44319-023-00026-0
Nat Commun. 2024 Jan 02. 15(1): 91

Experimental determination and mathematical modeling of standard shapes of forming autophagosomes.

Yuji Sakai, Satoru Takahashi, Ikuko Koyama-Honda, Chieko Saito, Noboru Mizushima.

The formation of autophagosomes involves dynamic morphological changes of a phagophore from a flat membrane cisterna into a cup-shaped intermediate and a spherical autophagosome. However, the physical mechanism behind these morphological changes remains elusive. Here, we determine the average shapes of phagophores by statistically investigating three-dimensional electron micrographs of more than 100 phagophores. The results show that the cup-shaped structures adopt a characteristic morphology; they are longitudinally elongated, and the rim is catenoidal with an outwardly recurved shape. To understand these characteristic shapes, we establish a theoretical model of the shape of entire phagophores. The model quantitatively reproduces the average morphology and reveals that the characteristic shape of phagophores is primarily determined by the relative size of the open rim to the total surface area. These results suggest that the seemingly complex morphological changes during autophagosome formation follow a stable path determined by elastic bending energy minimization.

DOI: https://doi.org/10.1038/s41467-023-44442-1
J Am Chem Soc. 2023 Dec 29.

Self-Sorting Collagen Heterotrimers.

Valdrin Islami, Philipp Bittner, Tomas Fiala, Nina B Hentzen, Renato Zenobi, Helma Wennemers.

Nature uses elaborate methods to control protein assembly, including that of heterotrimeric collagen. Here, we established design principles for the composition and register-selective assembly of synthetic collagen heterotrimers. The assembly code enabled the self-sorting of eight different strands into three─out of 512 possible─triple helices via complementary (4S)-aminoproline and aspartate residues. Native ESI-MS corroborated the specific assembly into coexisting heterotrimers.

DOI: https://doi.org/10.1021/jacs.3c12295
bioRxiv. 2023 Dec 15. pii: 2023.12.14.571662. [Epub ahead of print]

A proteolytic AAA+ machine poised to unfold a protein substrate.

Alireza Ghanbarpour, Robert T Sauer, Joseph H Davis.

AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.

DOI: https://doi.org/10.1101/2023.12.14.571662
Cell Rep. 2024 Jan 03. pii: S2211-1247(23)01648-0. [Epub ahead of print]43(1): 113637

TREX2 deficiency suppresses spontaneous and genotoxin-associated mutagenesis.

Teresa Marple, Mi Young Son, Xiaodong Cheng, Jun Ho Ko, Patrick Sung, Paul Hasty.

  TREX2, a 3'-5' exonuclease, is a part of the DNA damage tolerance (DDT) pathway that stabilizes replication forks (RFs) by ubiquitinating PCNA along with the ubiquitin E3 ligase RAD18 and other DDT factors. Mismatch repair (MMR) corrects DNA polymerase errors, including base mismatches and slippage. Here we demonstrate that TREX2 deletion reduces mutations in cells upon exposure to genotoxins, including those that cause base lesions and DNA polymerase slippage. Importantly, we show that TREX2 generates most of the spontaneous mutations in MMR-mutant cells derived from mice and people. TREX2-induced mutagenesis is dependent on the nuclease and DNA-binding attributes of TREX2. RAD18 deletion also reduces spontaneous mutations in MMR-mutant cells, albeit to a lesser degree. Inactivation of both MMR and TREX2 additively increases RF stalls, while it decreases DNA breaks, consistent with a synthetic phenotype.

Keywords:  CP: Molecular biology; DNA damage tolerance; TREX2; mismatch repair; replication fork maintenance

DOI:  https://doi.org/10.1016/j.celrep.2023.113637
Blood. 2024 Jan 03. pii: blood.2023022993. [Epub ahead of print]

Degraders Upgraded: The Rise of PROTACs in Haematological Malignancies.

Joshua Ml Casan, John F Seymour.

Targeted protein degradation (TPD) is a revolutionary approach to targeted therapy in hematological malignancies that potentially circumvents many constraints of existing small molecule inhibitors. Heterobifunctional proteolysis targets chimeras (PROTACs) are the leading TPD drug class, with numerous agents now in clinical trials for a range of blood cancers. PROTACs harness the cell intrinsic protein recycling infrastructure, the ubiquitin-proteasome system (UPS), to completely degrade target proteins. Distinct from targeted small molecule inhibitor therapies, PROTACs can eliminate critical but conventionally 'undruggable' targets, overcome resistance mechanisms to small molecule therapies and can improve tissue specificity and off-target toxicity. Orally bioavailable, PROTACs are not dependent on the occupancy driven pharmacology inherent to inhibitory therapeutics, facilitating sub-stoichiometric dosing that does not require an active or allosteric target binding site. Preliminary clinical data demonstrate promising therapeutic activity in heavily pre-treated populations and novel technology platforms are poised to exploit myriad permutations of PROTAC molecular design to enhance efficacy and targeting specificity. As the field rapidly progresses and various non-PROTAC TPD drug candidates emerge, this review explores the scientific and pre-clinical foundations of PROTACs and presents them within common clinical contexts. Additionally, we examine the latest findings from ongoing active PROTAC clinical trials.

DOI: https://doi.org/10.1182/blood.2023022993
Mol Cell. 2023 Dec 29. pii: S1097-2765(23)01022-5. [Epub ahead of print]

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

Damon A Hofman, Jorge Ruiz-Orera, Ian Yannuzzi, Rakesh Murugesan, Adam Brown, Karl R Clauser, Alexandra L Condurat, Jip T van Dinter, Sem A G Engels, Amy Goodale, Jasper van der Lugt, Tanaz Abid, Li Wang, Kevin N Zhou, Jayne Vogelzang, Keith L Ligon, Timothy N Phoenix, Jennifer A Roth, David E Root, Norbert Hubner, Todd R Golub, Pratiti Bandopadhayay, Sebastiaan van Heesch, John R Prensner.

  A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

Keywords:  CRISPR; Ribo-seq; cancer; gene dependency; lncRNAs; medulloblastoma; non-canonical ORFs; translational regulation; uORF

DOI:  https://doi.org/10.1016/j.molcel.2023.12.003
Life Sci Alliance. 2024 Mar;pii: e202302368. [Epub ahead of print]7(3):

Variable PD-1 glycosylation modulates the activity of immune checkpoint inhibitors.

Chih-Wei Chu, Tomislav Čaval, Frederico Alisson-Silva, Akshaya Tankasala, Christina Guerrier, Gregg Czerwieniec, Heinz Läubli, Flavio Schwarz.

Monoclonal antibodies targeting the immune checkpoint PD-1 have provided significant clinical benefit across a number of solid tumors, with differences in efficacy and toxicity profiles possibly related to their intrinsic molecular properties. Here, we report that camrelizumab and cemiplimab engage PD-1 through interactions with its fucosylated glycan. Using a combination of protein and cell glycoengineering, we demonstrate that the two antibodies bind preferentially to PD-1 with core fucose at the asparagine N58 residue. We then provide evidence that the concentration of fucosylated PD-1 in the blood of non-small-cell lung cancer patients varies across different stages of disease. This study illustrates how glycoprofiling of surface receptors and related circulating forms can inform the development of differentiated antibodies that discriminate glycosylation variants and achieve enhanced selectivity, and paves the way toward the implementation of personalized therapeutic approaches.

DOI: https://doi.org/10.26508/lsa.202302368