bims-proteo 2023-09-03 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒09‒03
39 papers selected by
Eric Chevet, INSERM

Co-chaperone BAG3 enters autophagic pathway via its interaction with microtubule associated protein 1 light chain 3 beta.
Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control.
Characterization of endoplasmic reticulum-associated degradation in the human fungal pathogen Candida albicans.
Activity-based profiling of cullin-RING E3 networks by conformation-specific probes.
The spatiotemporal control of ER membrane fragmentation during reticulophagy.
Membrane homeostasis beyond fluidity: control of membrane compressibility.
PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development.
DYRK3 enables secretory trafficking by maintaining the liquid-like state of ER exit sites.
Tube geometry controls protein cluster conformation and stability on the endoplasmic reticulum surface.
MARCH7-mediated ubiquitination decreases the solubility of ATG14 to inhibit autophagy.
Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network.
Targeted Degradation of Alpha-Synuclein by Autophagosome-Anchoring Chimera Peptides.
Systematic profiling of dominant ubiquitin variants reveals key functional nodes contributing to evolutionary selection.
NBR1-mediated selective chloroplast autophagy is important to plant stress tolerance.
Examining SRP pathway function in mRNA localization to the endoplasmic reticulum.
The α-crystallin chaperones undergo a quasi-ordered co-aggregation process in response to saturating client interaction.
Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A.
The transcription factor NRF1 (NFE2L1) activates aggrephagy by inducing p62 and GABARAPL1 after proteasome inhibition to maintain proteostasis.
Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT.
Deep and fast label-free Dynamic Organellar Mapping.
Pharmacophore-Based Machine Learning Model To Predict Ligand Selectivity for E3 Ligase Binders.
Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.
Nacα protects the larval fat body from cell death by maintaining cellular proteostasis in Drosophila.
Thioredoxin regulates the redox state and the activity of the human tRNA ligase complex.
Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response.
Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.
Comprehensive quantitative modeling of translation efficiency in a genome-reduced bacterium.
The unfolded protein response links ER stress to cancer-associated thrombosis.
The plant-unique protein DRIF1 coordinates with SNX1 to regulate membrane protein homeostasis.
SEL1L preserves CD8+ T-cell survival and homeostasis by fine-tuning PERK signaling and the IL-15 receptor-mediated mTORC1 axis.
Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.
Ubiquitination and deubiquitination: Implications on cancer therapy.
Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.
Global histone H2B degradation regulates insulin/IGF signaling-mediated nutrient stress.
Structure of the preholoproteasome reveals late steps in proteasome core particle biogenesis.
Inhibiting sorting nexin 10 promotes mucosal healing through SREBP2-mediated stemness restoration of intestinal stem cells.
In vitro Analysis of Stalled Ribosomes Using Puromycin Incorporation.
TOR-mediated Ypt1 phosphorylation regulates autophagy initiation complex assembly.
The E3/E4 ubiquitin ligase UFD-2 suppresses normal and oncogenic signaling mediated by a Raf ortholog in Caenorhabditis elegans.

Traffic. 2023 Sep 01.

Co-chaperone BAG3 enters autophagic pathway via its interaction with microtubule associated protein 1 light chain 3 beta.

Hagen Körschgen, Marius Baeken, Daniel Schmitt, Heike Nagel, Christian Behl.

  The co-chaperone BAG3 is a hub for a variety of cellular pathways via its multiple domains and its interaction with chaperones of the HSP70 family or small HSPs. During aging and under cellular stress conditions in particular, BAG3, together with molecular chaperones, ensures the sequestration of aggregated or aggregation-prone ubiquitinated proteins to the autophagic-lysosomal system via ubiquitin receptors. Accumulating evidence for BAG3-mediated selective autophagy independent of cargo ubiquitination led to analyses predicting a direct interaction of BAG3 with LC3 proteins. Phylogenetically, BAG3 comprises several highly conserved potential LIRs, LC3-interacting regions, which might allow for the direct targeting of BAG3 including its cargo to autophagosomes and drive their autophagic degradation. Based on pull-down experiments, peptide arrays and proximity ligation assays, our results provide evidence of an interaction of BAG3 with LC3B. In addition, we could demonstrate that disabling all predicted LIRs abolished the inducibility of a colocalization of BAG3 with LC3B-positive structures and resulted in a substantial decrease of BAG3 levels within purified native autophagic vesicles compared with wild-type BAG3. These results suggest an autophagic targeting of BAG3 via interaction with LC3B. Therefore, we conclude that, in addition to being a key co-chaperone to HSP70, BAG3 may also act as a cargo receptor for client proteins, which would significantly extend the role of BAG3 in selective macroautophagy and protein quality control.

Keywords:  BAG3; BAG3-mediated autophagy; Bcl2-associated athanogene 3; LC3; LIR

DOI:  https://doi.org/10.1111/tra.12916
Cell Rep. 2023 Aug 30. pii: S2211-1247(23)01067-7. [Epub ahead of print]42(9): 113056

Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control.

Lukas-Adrian Gurzeler, Marion Link, Yvonne Ibig, Isabel Schmidt, Olaf Galuba, Julian Schoenbett, Christelle Gasser-Didierlaurant, Christian N Parker, Xiaohong Mao, Francis Bitsch, Markus Schirle, Philipp Couttet, Frederic Sigoillot, Jana Ziegelmüller, Anne-Christine Uldry, Wojciech Teodorowicz, Niko Schmiedeberg, Oliver Mühlemann, Jürgen Reinhardt.

  Suppression of premature termination codons (PTCs) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two potent readthrough promoters-NVS1.1 and NVS2.1-that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, and activating a branch of the ribosome-associated quality control network, which involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25.

Keywords:  CFTR; CP: Molecular biology; E3 ligase; GCN1; Hurler syndrome; IDUA; RNF14; RNF25; RQC; cystic fibrosis; eRF1; proteasomal degradation; readthrough promoter; ribosome collisions; ubiquitination

DOI:  https://doi.org/10.1016/j.celrep.2023.113056
PeerJ. 2023 ;11 e15897

Characterization of endoplasmic reticulum-associated degradation in the human fungal pathogen Candida albicans.

Ellen M Doss, Joshua M Moore, Bryce H Harman, Emma H Doud, Eric M Rubenstein, Douglas A Bernstein.

  Background: Candida albicans is the most prevalent human fungal pathogen. In immunocompromised individuals, C. albicans can cause serious systemic disease, and patients infected with drug-resistant isolates have few treatment options. The ubiquitin-proteasome system has not been thoroughly characterized in C. albicans. Research from other organisms has shown ubiquitination is important for protein quality control and regulated protein degradation at the endoplasmic reticulum (ER) via ER-associated protein degradation (ERAD).Methods: Here we perform the first characterization, to our knowledge, of ERAD in a human fungal pathogen. We generated functional knockouts of C. albicans genes encoding three proteins predicted to play roles in ERAD, the ubiquitin ligases Hrd1 and Doa10 and the ubiquitin-conjugating enzyme Ubc7. We assessed the fitness of each mutant in the presence of proteotoxic stress, and we used quantitative tandem mass tag mass spectrometry to characterize proteomic alterations in yeast lacking each gene.
Results: Consistent with a role in protein quality control, yeast lacking proteins thought to contribute to ERAD displayed hypersensitivity to proteotoxic stress. Furthermore, each mutant displayed distinct proteomic profiles, revealing potential physiological ERAD substrates, co-factors, and compensatory stress response factors. Among candidate ERAD substrates are enzymes contributing to ergosterol synthesis, a known therapeutic vulnerability of C. albicans. Together, our results provide the first description of ERAD function in C. albicans, and, to our knowledge, any pathogenic fungus.

Keywords:  Candida albicans; Doa10; Endoplasmic reticulum-associated degradation; Hrd1; Mass spectrometry; Pathogenic fungi; Protein quality control; Ubc7; Ubiquitin ligase; Ubiquitin-conjugating enzyme

DOI:  https://doi.org/10.7717/peerj.15897
Nat Chem Biol. 2023 Aug 31.

Activity-based profiling of cullin-RING E3 networks by conformation-specific probes.

Lukas T Henneberg, Jaspal Singh, David M Duda, Kheewoong Baek, David Yanishevski, Peter J Murray, Matthias Mann, Sachdev S Sidhu, Brenda A Schulman.

The cullin-RING ubiquitin ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We developed a synthetic antibody recognizing the active conformation of NEDD8-linked cullins. Implementing the probe to profile cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing E3s revealed the complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, and prime efficiency of targeted protein degradation. Our probe also unveiled differential rewiring of CRL networks across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate the development of degrader drugs.

DOI: https://doi.org/10.1038/s41589-023-01392-5
Autophagy. 2023 Aug 31. 1-2

The spatiotemporal control of ER membrane fragmentation during reticulophagy.

Xinyi Wang, Boran Li, Qiming Sun.

  Reticulophagy is an evolutionarily conserved mechanism essential to maintain the endoplasmic reticulum (ER) homeostasis. A series of studies identified a panel of reticulophagy receptors. However, it remains unclear how these receptors sense upstream signals for spatiotemporal control of reticulophagy and how ER is fragmented into small pieces for sequestration into phagophores. Recently, we and others showed that the oligomerization of RETREG1/FAM134B (reticulophagy regulator 1), an reticulophagy receptor, triggers the scission of ER membrane to facilitate reticulophagy. Furthermore, we demonstrated that upstream signals are transduced by sequential phosphorylation and acetylation of RETREG1, which stimulate its oligomerization, ER fragmentation and reticulophagy. Our work provides further mechanistic insights into how reticulophagy receptor conveys cellular signals to fine-tune of ER homeostasis.Abbreviations: ER, endoplasmic reticulum; MAP1LC3, microtubule-associated protein light chain 3; RETREG1, reticulophagy regulator 1; RHD, reticulon-homology domain.

Keywords:  RETREG1; acetylation; membrane fragmentation; phosphorylation; reticulophagy

DOI:  https://doi.org/10.1080/15548627.2023.2252723
Trends Biochem Sci. 2023 Aug 29. pii: S0968-0004(23)00207-4. [Epub ahead of print]

Membrane homeostasis beyond fluidity: control of membrane compressibility.

Mike F Renne, Robert Ernst.

  Biomembranes are complex materials composed of lipids and proteins that compartmentalize biochemistry. They are actively remodeled in response to physical and metabolic cues, as well as during cell differentiation and stress. The concept of homeoviscous adaptation has become a textbook example of membrane responsiveness. Here, we discuss limitations and common misconceptions revolving around it. By highlighting key moments in the life cycle of a transmembrane protein, we illustrate that membrane thickness and a finely regulated membrane compressibility are crucial to facilitate proper membrane protein insertion, function, sorting, and inheritance. We propose that the unfolded protein response (UPR) provides a mechanism for endoplasmic reticulum (ER) membrane homeostasis by sensing aberrant transverse membrane stiffening and triggering adaptive responses that re-establish membrane compressibility.

Keywords:  homeoviscous adaptation; membrane compressibility; membrane fluidity; membrane thickness; unfolded protein response

DOI:  https://doi.org/10.1016/j.tibs.2023.08.004
Cell. 2023 Aug 21. pii: S0092-8674(23)00862-0. [Epub ahead of print]

PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development.

Ruoxi Wang, Tina M Fortier, Fei Chai, Guangyan Miao, James L Shen, Lucas J Restrepo, Jeromy J DiGiacomo, Panagiotis D Velentzas, Eric H Baehrecke.

  Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. Here, we describe a developmentally programmed selective ER clearance by autophagy. We show that Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy.

Keywords:  Drosophila; ER-phagy; Keap1; PINK1; Parkin; Rtnl1

DOI:  https://doi.org/10.1016/j.cell.2023.08.008
Dev Cell. 2023 Aug 24. pii: S1534-5807(23)00401-X. [Epub ahead of print]

DYRK3 enables secretory trafficking by maintaining the liquid-like state of ER exit sites.

Raffaella Gallo, Arpan Kumar Rai, Alexa B R McIntyre, Katrina Meyer, Lucas Pelkmans.

  The dual-specificity kinase DYRK3 controls the formation and dissolution of multiple biomolecular condensates, regulating processes including stress recovery and mitotic progression. Here, we report that DYRK3 functionally interacts with proteins associated with endoplasmic reticulum (ER) exit sites (ERESs) and that inhibition of DYRK3 perturbs the organization of the ERES-Golgi interface and secretory trafficking. DYRK3-mediated regulation of ERES depends on the N-terminal intrinsically disordered region (IDR) of the peripheral membrane protein SEC16A, which co-phase separates with ERES components to form liquid-like condensates on the surface of the ER. By modulating the liquid-like properties of ERES, we show that their physical state is essential for functional cargo trafficking through the early secretory pathway. Our findings support a mechanism whereby phosphorylation by DYRK3 and its reversal by serine-threonine phosphatases regulate the material properties of ERES to create a favorable physicochemical environment for directional membrane traffic in eukaryotic cells.

Keywords:  COPII; DYRK3; ERES; LLPS; SEC16A; kinase; material properties; optogenetics; phase separation; secretory pathway

DOI:  https://doi.org/10.1016/j.devcel.2023.08.005
Soft Matter. 2023 Aug 29.

Tube geometry controls protein cluster conformation and stability on the endoplasmic reticulum surface.

Liam T Kischuck, Aidan I Brown.

The endoplasmic reticulum (ER), a cellular organelle that forms a cell-spanning network of tubes and sheets, is an important location of protein synthesis and folding. When the ER experiences sustained unfolded protein stress, IRE1 proteins embedded in the ER membrane activate and assemble into clusters as part of the unfolded protein response (UPR). We use kinetic Monte Carlo simulations to explore IRE1 clustering dynamics on the surface of ER tubes. While initially growing clusters are approximately round, once a cluster is sufficiently large a shorter interface length can be achieved by 'wrapping' around the ER tube. A wrapped cluster can grow without further interface length increases. Relative to wide tubes, narrower tubes enable cluster wrapping at smaller cluster sizes. Our simulations show that wrapped clusters on narrower tubes grow more rapidly, evaporate more slowly, and require a lower protein concentration to grow compared to equal-area round clusters on wider tubes. These results suggest that cluster wrapping, facilitated by narrower tubes, could be an important factor in the growth and stability of IRE1 clusters and thus impact the persistence of the UPR, connecting geometry to signaling behavior. This work is consistent with recent experimental observations of IRE1 clusters wrapped around narrow tubes in the ER network.

DOI: https://doi.org/10.1039/d3sm00694h
Cell Rep. 2023 Aug 25. pii: S2211-1247(23)01056-2. [Epub ahead of print]42(9): 113045

MARCH7-mediated ubiquitination decreases the solubility of ATG14 to inhibit autophagy.

Xue Shi, Wenfeng Wu, Zhenhuan Feng, Peiyang Fan, Ruona Shi, Xiaofei Zhang.

  Autophagy is a fundamental biological process critical to all eukaryotic cellular life. Although autophagy has been increasingly studied, how its process is precisely coordinated remains an open question. ATG14 (ATG14L/Barkor) is known to play a crucial role in both autophagosome formation and autophagosome-lysosome fusion. However, how ATG14 is regulated, especially at the post-translation level, is still not clear. Here, we report that MARCH7 (membrane-associated ring-CH-type finger 7), an E3 ubiquitin ligase, inhibits autophagy by ubiquitinating ATG14. MARCH7 significantly promotes K6-, K11-, and K63-linked mixed polyubiquitination on ATG14, triggering the aggregation of ATG14 and reducing its solubility in cells. Functionally, we find that MARCH7 depletion decreases the number of aggresome-like induced structures (ALISs). Mechanistically, we show that ubiquitinated ATG14 has fewer interactions with STX17, leading to the inhibition of autophagy flux. Collectively, our study reveals a mechanism in regulating autophagy and suggests a potential strategy for the treatment of autophagy-related diseases.

Keywords:  CP: Cell biology

DOI:  https://doi.org/10.1016/j.celrep.2023.113045
Autophagy. 2023 Aug 30. 1-20

Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network.

Saurabh Srivastav, Kevin van der Graaf, Pratibha Singh, Alloysius Budi Utama, Matthew D Meyer, James A McNew, Michael Stern.

  ABBREVIATIONS: atl atlastin; ALR autophagic lysosome reformation; ER endoplasmic reticulum; GFP green fluorescent protein; HSP hereditary spastic paraplegia; Lamp1 lysosomal associated membrane protein 1 PolyUB polyubiquitin; RFP red fluorescent protein; spin spinster; mTor mechanistic Target of rapamycin; VCP valosin containing protein.

Keywords:  ATG8; Hereditary Spastic Paraplegia; LAMP1; Neurodegeneration; endosome; protein aggregates; rab7

DOI:  https://doi.org/10.1080/15548627.2023.2249794
J Med Chem. 2023 Aug 31.

Targeted Degradation of Alpha-Synuclein by Autophagosome-Anchoring Chimera Peptides.

Yichen Tong, Wentao Zhu, Jian Chen, Wenqian Zhang, Fang Xu, Jiyan Pang.

Targeted protein degradation (TPD) confers knockdown of "undruggable" targets such as alpha-synuclein (αSyn), a pathogenic protein in multiple neurodegenerative diseases. Though many of these proteins were mainly degraded through the autophagy-lysosome pathway (ALP), few TPD tools harnessing the ALP were reported. Herein, we developed a strategy termed autophagosome-anchoring chimera (ATACC), in which the protein of interest (POI) can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of an LC3-interacting region (LIR)-containing bifunctional peptide, and the selective autophagy of the POI is thus facilitated. A series of αSyn-targeting ATACC peptides were designed and synthesized. Biological evaluations demonstrated that these compounds could degrade αSyn specifically and effectively through a "chemical-induced cargo recognition-ALP degradation" mechanism. The neuroprotective effects of ATACC peptide P1 were further validated in vitro and in vivo. Collectively, our results provided a new TPD tool and revealed a potential therapeutic strategy against synucleinopathies.

DOI: https://doi.org/10.1021/acs.jmedchem.3c01303
Cell Rep. 2023 Aug 31. pii: S2211-1247(23)01075-6. [Epub ahead of print]42(9): 113064

Systematic profiling of dominant ubiquitin variants reveals key functional nodes contributing to evolutionary selection.

Amrita Arpita Padhy, David Mavor, Subhashree Sahoo, Daniel N A Bolon, Parul Mishra.

  Dominant-negative mutations can help to investigate the biological mechanisms and to understand the selective pressures for multifunctional proteins. However, most studies have focused on recessive mutant effects that occur in the absence of a second functional gene copy, which overlooks the fact that most eukaryotic genomes contain more than one copy of many genes. We have identified dominant effects on yeast growth rate among all possible point mutations in ubiquitin expressed alongside a wild-type allele. Our results reveal more than 400 dominant-negative mutations, indicating that dominant-negative effects make a sizable contribution to selection acting on ubiquitin. Cellular and biochemical analyses of individual ubiquitin variants show that dominant-negative effects are explained by varied accumulation of polyubiquitinated cellular proteins and/or defects in conjugation of ubiquitin variants to ubiquitin ligases. Our approach to identify dominant-negative mutations is general and can be applied to other proteins of interest.

Keywords:  CP: Cell biology; EMPIRIC; dominant-negative mutations; mutants; overexpression scan; polyubiquitination; toxic variants; ubiquitin; ubiquitin evolution; yeast growth effects

DOI:  https://doi.org/10.1016/j.celrep.2023.113064
Autophagy. 2023 Aug 27. 1-2

NBR1-mediated selective chloroplast autophagy is important to plant stress tolerance.

Hui Zhang, Qihua Ling.

  Macroautophagy/autophagy is a conserved process in eukaryotes responsible for degrading unwanted or damaged macromolecules and organelles through the lysosome or vacuole for recycling and reutilization. Our previous studies revealed the degradation of chloroplast proteins through a pathway dependent on the ubiquitin proteasome system, known as CHLORAD. Recently, we demonstrated a role for selective autophagy in regulating chloroplast protein import and enhancing stress tolerance in plants. Specifically, we found that K63-ubiquitination of TOC components at the chloroplast outer envelope membrane is recognized by the selective autophagy adaptor NBR1, leading to the degradation of TOC proteins under UV-B irradiation and heat stresses in Arabidopsis. This process was shown to control chloroplast protein import and influence photosynthetic activity. Based on our results, we have, for the first time, demonstrated that selective autophagy plays a vital role in chloroplast protein degradation, specifically in response to certain abiotic stresses.

Keywords:  Arabidopsis thaliana; NBR1; chloroplast; protein import; selective autophagy

DOI:  https://doi.org/10.1080/15548627.2023.2251324
RNA. 2023 Aug 29. pii: rna.079643.123. [Epub ahead of print]

Examining SRP pathway function in mRNA localization to the endoplasmic reticulum.

Jessica Rae Child, Alex C Hofler, Qiang Chen, Brenda H Yang, JohnCarlo Kristofich, Tianli Zheng, Molly M Hannigan, Andrew L Elles, David W Reid, Christopher V Nicchitta.

  Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well-established; its role in RNA localization to the ER remains however unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding alpha subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SRP receptor (SR) knockout mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SRP receptor expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER..

Keywords:  RNA localization; SRP receptor; endoplasmic reticulum; mRNA; ribosome

DOI:  https://doi.org/10.1261/rna.079643.123
bioRxiv. 2023 Aug 17. pii: 2023.08.15.553435. [Epub ahead of print]

The α-crystallin chaperones undergo a quasi-ordered co-aggregation process in response to saturating client interaction.

Adam P Miller, Susan E O'Neill, Kirsten J Lampi, Steve L Reichow.

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. This study investigates structural changes in αAc and αBc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined αAc and αBc in their apo-states and at various stages of client-induced co-aggregation, using lysozyme as a model client. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP scaffold. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP scaffolding with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across both αAc and αBc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of sHSPs, carrying potential implications for a pathway toward cataract formation.

DOI: https://doi.org/10.1101/2023.08.15.553435
Mol Cell. 2023 Aug 17. pii: S1097-2765(23)00607-X. [Epub ahead of print]

Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A.

Zhiheng Deng, Huasong Ai, Maoshen Sun, Zebin Tong, Yunxiang Du, Qian Qu, Liying Zhang, Ziyu Xu, Shixian Tao, Qiang Shi, Jia-Bin Li, Man Pan, Lei Liu.

  Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.

Keywords:  Bre1-Rad6; RNF20/RNF40-hRAD6A; chemical biology; cryo-electron microscopy; epigenetics; histone H2B monoubiquitylation; structural biology; ubiquitylation mechanism

DOI:  https://doi.org/10.1016/j.molcel.2023.08.001
Sci Rep. 2023 Sep 01. 13(1): 14405

The transcription factor NRF1 (NFE2L1) activates aggrephagy by inducing p62 and GABARAPL1 after proteasome inhibition to maintain proteostasis.

Atsushi Hatanaka, Sota Nakada, Gen Matsumoto, Katsuya Satoh, Iori Aketa, Akira Watanabe, Tomoaki Hirakawa, Tadayuki Tsujita, Tsuyoshi Waku, Akira Kobayashi.

The ubiquitin‒proteasome system (UPS) and autophagy are the two primary cellular pathways of misfolded or damaged protein degradation that maintain cellular proteostasis. When the proteasome is dysfunctional, cells compensate for impaired protein clearance by activating aggrephagy, a type of selective autophagy, to eliminate ubiquitinated protein aggregates; however, the molecular mechanisms by which impaired proteasome function activates aggrephagy remain poorly understood. Here, we demonstrate that activation of aggrephagy is transcriptionally induced by the transcription factor NRF1 (NFE2L1) in response to proteasome dysfunction. Although NRF1 has been previously shown to induce the expression of proteasome genes after proteasome inhibition (i.e., the proteasome bounce-back response), our genome-wide transcriptome analyses identified autophagy-related p62/SQSTM1 and GABARAPL1 as genes directly targeted by NRF1. Intriguingly, NRF1 was also found to be indispensable for the formation of p62-positive puncta and their colocalization with ULK1 and TBK1, which play roles in p62 activation via phosphorylation. Consistently, NRF1 knockdown substantially reduced the phosphorylation rate of Ser403 in p62. Finally, NRF1 selectively upregulated the expression of GABARAPL1, an ATG8 family gene, to induce the clearance of ubiquitinated proteins. Our findings highlight the discovery of an activation mechanism underlying NRF1-mediated aggrephagy through gene regulation when proteasome activity is impaired.

DOI: https://doi.org/10.1038/s41598-023-41492-9
Nat Struct Mol Biol. 2023 Aug 31.

Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT.

Eva Absmeier, Viswanathan Chandrasekaran, Francis J O'Reilly, James A W Stowell, Juri Rappsilber, Lori A Passmore.

Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4-NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4-NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4-NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.

DOI: https://doi.org/10.1038/s41594-023-01075-8
Nat Commun. 2023 Aug 29. 14(1): 5252

Deep and fast label-free Dynamic Organellar Mapping.

Julia P Schessner, Vincent Albrecht, Alexandra K Davies, Pavel Sinitcyn, Georg H H Borner.

The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.

DOI: https://doi.org/10.1038/s41467-023-41000-7
ACS Omega. 2023 Aug 22. 8(33): 30177-30185

Pharmacophore-Based Machine Learning Model To Predict Ligand Selectivity for E3 Ligase Binders.

Reagon Karki, Yojana Gadiya, Philip Gribbon, Andrea Zaliani.

E3 ligases are enzymes that play a critical role in ubiquitin-mediated protein degradation and are involved in various cellular processes. Pharmacophore analysis is a useful approach for predicting E3 ligase binding selectivity, which involves identifying key chemical features necessary for a ligand to interact with a specific protein target cavity. While pharmacophore analysis is not always sufficient to accurately predict ligand binding affinity, it can be a valuable tool for filtering and/or designing focused libraries for screening campaigns. In this study, we present a fast and an inexpensive approach using a pharmacophore fingerprinting scheme known as ErG, which is used in a multi-class machine learning classification model. This model can assign the correct E3 ligase binder to its known E3 ligase and predict the probability of each molecule to bind to different E3 ligases. Practical applications of this approach are demonstrated on commercial libraries such as Asinex for the rational design of E3 ligase binders. The scripts and data associated with this study can be found on GitHub at https://github.com/Fraunhofer-ITMP/E3_binder_Model.

DOI: https://doi.org/10.1021/acsomega.3c02803
ACS Chem Biol. 2023 Aug 29.

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.

Jin Gan, Jelle de Vries, Jimmy J L L Akkermans, Yassene Mohammed, Rayman T N Tjokrodirijo, Arnoud H de Ru, Robbert Q Kim, David A Vargas, Vito Pol, Rudi Fasan, Peter A van Veelen, Jacques Neefjes, Hans van Dam, Huib Ovaa, Aysegul Sapmaz, Paul P Geurink.

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.

DOI: https://doi.org/10.1021/acschembio.3c00227
Nat Commun. 2023 Sep 01. 14(1): 5328

Nacα protects the larval fat body from cell death by maintaining cellular proteostasis in Drosophila.

Takayuki Yamada, Yuto Yoshinari, Masayuki Tobo, Okiko Habara, Takashi Nishimura.

Protein homeostasis (proteostasis) is crucial for the maintenance of cellular homeostasis. Impairment of proteostasis activates proteotoxic and unfolded protein response pathways to resolve cellular stress or induce apoptosis in damaged cells. However, the responses of individual tissues to proteotoxic stress and evoking cell death program have not been extensively explored in vivo. Here, we show that a reduction in Nascent polypeptide-associated complex protein alpha subunit (Nacα) specifically and progressively induces cell death in Drosophila fat body cells. Nacα mutants disrupt both ER integrity and the proteasomal degradation system, resulting in caspase activation through JNK and p53. Although forced activation of the JNK and p53 pathways was insufficient to induce cell death in the fat body, the reduction of Nacα sensitized fat body cells to intrinsic and environmental stresses. Reducing overall protein synthesis by mTor inhibition or Minute mutants alleviated the cell death phenotype in Nacα mutant fat body cells. Our work revealed that Nacα is crucial for protecting the fat body from cell death by maintaining cellular proteostasis, thus demonstrating the coexistence of a unique vulnerability and cell death resistance in the fat body.

DOI: https://doi.org/10.1038/s41467-023-41103-1
RNA. 2023 Aug 30. pii: rna.079732.123. [Epub ahead of print]

Thioredoxin regulates the redox state and the activity of the human tRNA ligase complex.

Dhaarsini Jaksch, Johanna Irnstorfer, Petra-Franziska Kalman, Javier Martinez.

The mammalian tRNA ligase complex (tRNA-LC) catalyzes the splicing of intron-containing pre-tRNAs in the nucleus and the splicing of XBP1 mRNA during the unfolded protein response (UPR) in the cytoplasm. We recently reported that the tRNA-LC co-evolved with PYROXD1, an essential oxidoreductase that protects the catalytic cysteine of RTCB, the catalytic subunit of the tRNA-LC, against aerobic oxidation. In this study we show that the oxidoreductase Thioredoxin (TRX) preserves the enzymatic activity of RTCB under otherwise inhibiting concentrations of oxidants. TRX physically interacts with oxidized RTCB, and reduces and re-activates RTCB through the action of its redox-active cysteine pair. We further show that TRX interacts with RTCB at late stages of UPR. Since the interaction requires oxidative conditions, our findings suggest that prolonged UPR generates reactive oxygen species. Thus, our results support a functional role for TRX in securing and repairing the active site of the tRNA-LC, thereby allowing pre-tRNA splicing and UPR to occur when cells encounter mild, but still inhibitory levels of reactive oxygen species.

DOI: https://doi.org/10.1261/rna.079732.123
Mol Cell. 2023 Aug 24. pii: S1097-2765(23)00640-8. [Epub ahead of print]

Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response.

Robert V Swanda, Quanquan Ji, Xincheng Wu, Jingyue Yan, Leiming Dong, Yuanhui Mao, Saori Uematsu, Yizhou Dong, Shu-Bing Qian.

  The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.

Keywords:  AhR; cancer therapy; cysteine; cystine; ferroptosis; lysosome; mRNA; nutrient stress

DOI:  https://doi.org/10.1016/j.molcel.2023.08.004
Cell Rep. 2023 Aug 31. pii: S2211-1247(23)01090-2. [Epub ahead of print]42(9): 113079

Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

Marwa M Afifi, Adrijana Crncec, James A Cornwell, Christophe Cataisson, Debasish Paul, Laila M Ghorab, Maria O Hernandez, Madeline Wong, Noemi Kedei, Steven D Cappell.

  Cells can irreversibly exit the cell cycle and become senescent to safeguard against uncontrolled proliferation. While the p53-p21 and p16-Rb pathways are thought to mediate senescence, they also mediate reversible cell cycle arrest (quiescence), raising the question of whether senescence is actually reversible or whether alternative mechanisms underly the irreversibility associated with senescence. Here, we show that senescence is irreversible and that commitment to and maintenance of senescence are mediated by irreversible MYC degradation. Senescent cells start dividing when a non-degradable MYC mutant is expressed, and quiescent cells convert to senescence when MYC is knocked down. In early oral carcinogenesis, epithelial cells exhibit MYC loss and become senescent as a safeguard against malignant transformation. Later stages of oral premalignant lesions exhibit elevated MYC levels and cellular dysplasia. Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.

Keywords:  CDK4/6; CP: Cell biology; MEK; MYC; cell cycle; palbociclib; pre-malignant lesions; senescence; time-lapse imaging; trametinib

DOI:  https://doi.org/10.1016/j.celrep.2023.113079
Mol Syst Biol. 2023 Aug 29. e11301

Comprehensive quantitative modeling of translation efficiency in a genome-reduced bacterium.

Marc Weber, Adrià Sogues, Eva Yus, Raul Burgos, Carolina Gallo, Sira Martínez, Maria Lluch-Senar, Luis Serrano.

  Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half-lives, proteins, and protein half-lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160-fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100-fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.

Keywords:  Mycoplasma pneumoniae; data integration; protein synthesis; ribosome profiling; translation efficiency

DOI:  https://doi.org/10.15252/msb.202211301
JCI Insight. 2023 Aug 31. pii: e170148. [Epub ahead of print]

The unfolded protein response links ER stress to cancer-associated thrombosis.

Oluwatoyosi Muse, Rushad Patell, Christian G Peters, Moua Yang, Emale El-Darzi, Sol Schulman, Anna Falanga, Marina Marchetti, Laura Russo, Jeffrey I Zwicker, Robert Flaumenhaft.

  Thrombosis is a common complication of advanced cancer. Yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasmas from gastric and non-small cell lung cancer patients who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasmas of patients who developed clots compared to those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors IRE1α or PERK. Release of extracellular vesicles bearing tissue factor (EVTF) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase TF synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by Arf1 knockdown or GBF1 antagonism, confirming the role of vesicular trafficking. Our findings show that UPR activation results in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.

Keywords:  Cancer; Hematology; Thrombosis

DOI:  https://doi.org/10.1172/jci.insight.170148
Plant Cell. 2023 Aug 30. pii: koad227. [Epub ahead of print]

The plant-unique protein DRIF1 coordinates with SNX1 to regulate membrane protein homeostasis.

Ying Zhu, Qiong Zhao, Wenhan Cao, Shuxian Huang, Changyang Ji, Wenxin Zhang, Marco Trujillo, Jinbo Shen, Liwen Jiang.

Membrane protein homeostasis is fine-tuned by the cellular pathways for vacuolar degradation and recycling, which ultimately facilitate plant growth and cell-environment interactions. The endosomal sorting complex required for transport (ESCRT) machinery plays important roles in regulating intraluminal vesicle (ILV) formation and membrane protein sorting to vacuoles. We previously showed that the plant-specific ESCRT component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FREE1) performs multiple functions in plants, although the underlying mechanisms remain elusive. In this study, we performed a suppressor screen of the FREE1-RNAi mutant and identified and characterized two suppressor of free1 (sof) mutants in Arabidopsis (Arabidopsis thaliana). These mutants, sof10 and sof641, result in a premature stop codon or a missense mutation in AT5G10370, respectively. This gene was named DEAH and RING domain-containing protein as FREE1 suppressor 1 (DRIF1). DRIF1 has a homologous gene, DRIF2, in the Arabidopsis genome with 95% identity to DRIF1. The embryos of drif1 drif2 mutants arrested at the globular stage and formed enlarged multivesicular bodies (MVBs) with an increased number of ILVs. DRIF1 is a membrane-associated protein that coordinates with retromer component sorting nexin 1 (SNX1) to regulate PIN-FORMED2 (PIN2) recycling to the plasma membrane. Altogether, our data demonstrate that DRIF1 is a unique retromer interactor that orchestrates FREE1-mediated ILV formation of MVBs and vacuolar sorting of membrane proteins for degradation in plants.

DOI: https://doi.org/10.1093/plcell/koad227
Cell Mol Immunol. 2023 Aug 30.

SEL1L preserves CD8+ T-cell survival and homeostasis by fine-tuning PERK signaling and the IL-15 receptor-mediated mTORC1 axis.

Yafeng Gao, Wenhui Li, Zhenghao Wang, Cangang Zhang, Yaping He, Xiaowei Liu, Kexin Tang, Weiguo Zhang, Qiaoming Long, Yong Liu, Jinping Zhang, Baojun Zhang, Lianjun Zhang.

  SEL1L-mediated endoplasmic reticulum-associated degradation (ERAD) plays critical roles in controlling protein homeostasis by degrading misfolded or terminal unfolded proteins. However, it remains unclear how SEL1L regulates peripheral T-cell survival and homeostasis. Herein, we found that SEL1L deficiency led to a greatly reduced frequency and number of mature T cells, which was further validated by adoptive transfer experiments or bone marrow chimera experiments, accompanied by the induction of multiple forms of cell death. Furthermore, SEL1L deficiency selectively disrupted naïve CD8+ T-cell homeostasis, as indicated by the severe loss of the naïve T-cell subset but an increase in the memory T-cell subset. We also found that SEL1L deficiency fueled mTORC1/c-MYC activation and induced a metabolic shift, which was largely attributable to enhanced expression of the IL-15 receptor α and β chains. Mechanistically, single-cell transcriptomic profiling and biochemical analyses further revealed that Sel1l-/- CD8+ T cells harbored excessive ER stress, particularly aberrant activation of the PERK-ATF4-CHOP-Bim pathway, which was alleviated by supplementing IL-7 or IL-15. Importantly, PERK inhibition greatly resolved the survival defects of Sel1l-/- CD8+ T cells. In addition, IRE1α deficiency decreased mTORC1 signaling in Sel1l-/- naïve CD8+ T cells by downregulating the IL-15 receptor α chain. Altogether, these observations suggest that the ERAD adaptor molecule SEL1L acts as an important checkpoint for preserving the survival and homeostasis of peripheral T cells by regulating the PERK signaling cascade and IL-15 receptor-mediated mTORC1 axis.

Keywords:  ER stress response; Endoplasmic reticulum-associated degradation; IRE1α; PERK; T-cell homeostasis

DOI:  https://doi.org/10.1038/s41423-023-01078-x
bioRxiv. 2023 Aug 22. pii: 2023.08.17.551257. [Epub ahead of print]

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

Alexander Kapustin, Sofia Serena Tsakali, Meredith Whitehead, George Chennell, Meng-Ying Wu, Chris Molenaar, Anton Kutikhin, Leo Bogdanov, Maxim Sinitsky, Kseniya Rubina, Aled Clayton, Frederik J Verweij, Dirk Michiel Pegtel, Simona Zingaro, Arseniy Lobov, Bozhana Zainullina, Dylan Owen, Maddy Parsons, Richard E Cheney, Derek Warren, Martin James Humphries, Thomas Iskratsch, Mark Holt, Catherine M Shanahan.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the β1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo . Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.Abstract Figure:

DOI: https://doi.org/10.1101/2023.08.17.551257
Biochim Biophys Acta Gene Regul Mech. 2023 Aug 24. pii: S1874-9399(23)00074-3. [Epub ahead of print] 194979

Ubiquitination and deubiquitination: Implications on cancer therapy.

Gunjan Dagar, Rakesh Kumar, Kamlesh K Yadav, Mayank Singh, Tej K Pandita.

  The ubiquitin proteasomal system (UPS) represents a highly regulated protein degradation pathway essential for maintaining cellular homeostasis. This system plays a critical role in several cellular processes, which include DNA damage repair, cell cycle checkpoint control, and immune response regulation. Recently, the UPS has emerged as a promising target for cancer therapeutics due to its involvement in oncogenesis and tumor progression. Here we aim to summarize the key aspects of the UPS and its significance in cancer therapeutics. We begin by elucidating the fundamental components of the UPS, highlighting the role of ubiquitin, E1-E3 ligases, and the proteasome in protein degradation. Furthermore, we discuss the intricate process of ubiquitination and proteasomal degradation, emphasizing the specificity and selectivity achieved through various signaling pathways. The dysregulation of the UPS has been implicated in cancer development and progression. Aberrant ubiquitin-mediated degradation of key regulatory proteins, such as tumor suppressors and oncoproteins, can lead to uncontrolled cell proliferation, evasion of apoptosis, and metastasis. We outline the pivotal role of the UPS in modulating crucial oncogenic pathways, including the regulation of cyclins, transcription factors, Replication stress components and DNA damage response. The increasing recognition of the UPS as a target for cancer therapeutics has spurred the development of small molecules, peptides, and proteasome inhibitors with the potential to restore cellular balance and disrupt tumor growth. We provide an overview of current therapeutic strategies aimed at exploiting the UPS, including the use of proteasome inhibitors, deubiquitinating enzyme inhibitors, and novel E3 ligase modulators. We further discuss novel emerging strategies for the development of next-generation drugs that target proteasome inhibitors. Exploiting the UPS for cancer therapeutics offers promising avenues for developing innovative and effective treatment strategies, providing hope for improved patient outcomes in the fight against cancer.

Keywords:  DNA damage; Deubiquitin enzymes (DUBs); Ubiquitin ligase; Ubiquitin proteasomal system (UPS)

DOI:  https://doi.org/10.1016/j.bbagrm.2023.194979
J Clin Invest. 2023 Aug 29. pii: e163591. [Epub ahead of print]

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

Efren A Reyes, David Castillo-Azofeifa, Jérémie Rispal, Tomas Wald, Rachel K Zwick, Brisa Palikuqi, Angela Mujukian, Shervin Rabizadeh, Alexander R Gupta, James M Gardner, Dario Boffelli, Zev J Gartner, Ophir D Klein.

  The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and non-inflamed samples from Inflammatory Bowel Disease (IBD) patients undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF-CFTR signaling axis in the adult intestine and identify epithelial-derived TNF as an upstream regulator of mucin homeostasis.

Keywords:  Cytokines; Epithelial transport of ions and water; Gastroenterology; Molecular genetics

DOI:  https://doi.org/10.1172/JCI163591
EMBO J. 2023 Aug 29. e113328

Global histone H2B degradation regulates insulin/IGF signaling-mediated nutrient stress.

Zhiwen Zhu, Dongdong Li, Zeran Jia, Wenhao Zhang, Yuling Chen, Ruixue Zhao, Yan-Ping Zhang, Wen-Hong Zhang, Haiteng Deng, Yinqing Li, Wei Li, Shouhong Guang, Guangshuo Ou.

  Eukaryotic organisms adapt to environmental fluctuations by altering their epigenomic landscapes and transcriptional programs. Nucleosomal histones carry vital epigenetic information and regulate gene expression, yet the mechanisms underlying chromatin-bound histone exchange remain elusive. Here, we found that histone H2Bs are globally degraded in Caenorhabditis elegans during starvation. Our genetic screens identified mutations in ubiquitin and ubiquitin-related enzymes that block H2B degradation in starved animals, identifying lysine 31 as the crucial residue for chromatin-bound H2B ubiquitination and elimination. Retention of aberrant nucleosomal H2B increased the association of the FOXO transcription factor DAF-16 with chromatin, generating an ectopic gene expression profile detrimental to animal viability when insulin/IGF signaling was reduced in well-fed animals. Furthermore, we show that the ubiquitin-proteasome system regulates chromosomal histone turnover in human cells. During larval development, C. elegans epidermal cells undergo H2B turnover after fusing with the epithelial syncytium. Thus, histone degradation may be a widespread mechanism governing dynamic changes of the epigenome.

Keywords:  Histone degradation; Insulin/IGF signaling; Ubiquitination

DOI:  https://doi.org/10.15252/embj.2022113328
Nat Struct Mol Biol. 2023 Aug 31.

Structure of the preholoproteasome reveals late steps in proteasome core particle biogenesis.

Richard M Walsh, Shaun Rawson, Helena M Schnell, Benjamin Velez, Tamayanthi Rajakumar, John Hanna.

Assembly of the proteasome's core particle (CP), a barrel-shaped chamber of four stacked rings, requires five chaperones and five subunit propeptides. Fusion of two half-CP precursors yields a complete structure but remains immature until active site maturation. Here, using Saccharomyces cerevisiae, we report a high-resolution cryogenic electron microscopy structure of preholoproteasome, a post-fusion assembly intermediate. Our data reveal how CP midline-spanning interactions induce local changes in structure, facilitating maturation. Unexpectedly, we find that cleavage may not be sufficient for propeptide release, as residual interactions with chaperones such as Ump1 hold them in place. We evaluated previous models proposing that dynamic conformational changes in chaperones drive CP fusion and autocatalytic activation by comparing preholoproteasome to pre-fusion intermediates. Instead, the data suggest a scaffolding role for the chaperones Ump1 and Pba1/Pba2. Our data clarify key aspects of CP assembly, suggest that undiscovered mechanisms exist to explain CP fusion/activation, and have relevance for diseases of defective CP biogenesis.

DOI: https://doi.org/10.1038/s41594-023-01081-w
Sci Adv. 2023 Sep;9(35): eadh5016

Inhibiting sorting nexin 10 promotes mucosal healing through SREBP2-mediated stemness restoration of intestinal stem cells.

Weilian Bao, Yan You, Jiahui Ni, Hui Hou, Jiaren Lyu, Guize Feng, Yirui Wang, Keyuan You, Sulin Zhang, Lijie Zhang, Xinyue Cao, Xu Wang, Haidong Li, Hong Li, Jiake Xu, Chenying Liu, Xiaomin Luo, Peng Du, Daofeng Chen, Xiaoyan Shen.

Intestinal stem cell (ISC) is a promising therapeutic target for inflammatory bowel disease. Cholesterol availability is critical for ISC stemness. Low plasma cholesterol is a typical feature of Crohn's disease (CD); however, its impact on mucosal healing remains unclear. Here, we identified an essential role of sorting nexin 10 (SNX10) in maintaining the stemness of ISCs. SNX10 expression in intestinal tissues positively correlates with the severity of human CD and mouse colitis. Conditional SNX10 knockout in intestinal epithelial cells or ISCs promotes intestinal mucosal repair by maintaining the ISC population associated with increased intracellular cholesterol synthesis. Disassociation of ERLIN2 with SCAP by SNX10 deletion enhances the activation of SREBP2, resulting in increased cholesterol biosynthesis. DC-SX029, a small-molecule inhibitor of SNX10, was used to verify the druggable potential of SNX10 for the treatment of patients with CD. Our study provides a strategy for mucosal healing through SREBP2-mediated stemness restoration of ISCs.

DOI: https://doi.org/10.1126/sciadv.adh5016
Bio Protoc. 2023 Aug 20. 13(16): e4744

In vitro Analysis of Stalled Ribosomes Using Puromycin Incorporation.

MaKenzie R Scarpitti, Michael G Kearse.

  Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via a ribosome-binding protein (e.g., FMRP). Stalled ribosomes can act as physical roadblocks for trailing ribosomes and ultimately can cause ribosome collisions that stimulate no-go mRNA decay. Detecting stalled or slowed ribosomes in cells by ribosome footprint profiling or classic polysome profiling is laborious, technically challenging, and low throughput. Here, we present a protocol to assay for stalled ribosomes on in vitro-transcribed reporter mRNAs using a robust, commercially available mammalian in vitro translation lysate and an optimized low-speed sucrose cushion. In short, we take advantage of the ability of puromycin to incorporate into the nascent polypeptide and cause the ribosome to dissociate from the mRNA during active elongation, as well as the ability to selectively pellet ribosomes through a low-speed sucrose cushion due to their large molecular weight. Stalled ribosomes are not actively elongating and do not incorporate puromycin, allowing the ribosome-bound mRNA to pellet in the low-speed sucrose cushion. RT-qPCR is used to quantify the amount of ribosome-bound reporter mRNA in the pellet. This workflow allows for direct assessment of stalled ribosomes and is fully amendable to insertion of putative stalling motifs in the target mRNA, as well as supplementation with recombinant proteins or small molecule inhibitors that target translation elongation. Key features This protocol is optimized for cap-dependent in vitro translation in the dynamic linear range. Details for generating capped reporter mRNA in one day are provided. Requires as little as one day to complete if starting with in vitro-transcribed mRNA. This protocol requires access to an ultracentrifuge and a real-time PCR system.

Keywords:  Cycloheximide; Elongation inhibition; Polysomes; Protein synthesis; Sucrose cushion; Ultracentrifugation

DOI:  https://doi.org/10.21769/BioProtoc.4744
EMBO J. 2023 Aug 28. e112814

TOR-mediated Ypt1 phosphorylation regulates autophagy initiation complex assembly.

Weijing Yao, Yuting Chen, Yingcong Chen, Pengwei Zhao, Jing Liu, Yi Zhang, Qiang Jiang, Choufei Wu, Yu Xie, Siyu Fan, Miao Ye, Yigang Wang, Yuyao Feng, Xue Bai, Mingzhu Fan, Shan Feng, Juan Wang, Yixian Cui, Hongguang Xia, Cheng Ma, Zhiping Xie, Liqin Zhang, Qiming Sun, Wei Liu, Cong Yi.

  The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1S174D phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.

Keywords:  ATG proteins; TOR; Ypt1; autophagy; stepwise assembly

DOI:  https://doi.org/10.15252/embj.2022112814
Sci Signal. 2023 Aug 29. 16(800): eabq4355

The E3/E4 ubiquitin ligase UFD-2 suppresses normal and oncogenic signaling mediated by a Raf ortholog in Caenorhabditis elegans.

Robert Townley, Augustin Deniaud, Kennedy S Stacy, Claudia S Rodriguez Torres, Fatemeh Cheraghi, Nicole B Wicker, Claire C de la Cova.

Signaling by the kinase cascade composed of Raf, MEK, and ERK is critical for animal development and is often inappropriately activated in human malignancies. We sought to identify factors that control signaling mediated by the Caenorhabditis elegans Raf ortholog LIN-45. A genetic screen showed that the degradation of LIN-45 required the E3/E4 ubiquitin ligase UFD-2. Both UFD-2 and its partner, the ATP-dependent segregase CDC-48, were required for the developmental regulation of LIN-45 protein abundance. We showed that UFD-2 acted in the same pathway as the E3 ubiquitin ligase SCFSEL-10 to decrease LIN-45 abundance in cells in which Raf-MEK-ERK signaling was most highly active. UFD-2 also reduced the protein abundance of activated LIN-45 carrying a mutation equivalent to the cancer-associated BRAF(V600E) variant. Our structure-function studies showed that the disruption of LIN-45 domains that mediate protein-protein interactions, including the conserved cysteine-rich domain and 14-3-3 binding motifs, were required for UFD-2-independent degradation of LIN-45. We propose a model in which UFD-2 and CDC-48 act downstream of SCFSEL-10 to remove LIN-45 from its protein interaction partners and facilitate proteasomal targeting and degradation. These findings imply that UFD-2 and CDC-48 may be important for Raf degradation during normal and oncogenic Ras and MAPK signaling in mammalian cells.

DOI: https://doi.org/10.1126/scisignal.abq4355