bims-proteo 2023-01-15 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–01–15
thirty-one papers selected by
Eric Chevet, INSERM

The ERAD system is restricted by elevated ceramides.
An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes.
Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.
The cellular pathways that maintain the quality control and transport of diverse potassium channels.
The unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family.
HSP70-binding motifs function as protein quality control degrons.
Zinc-finger protein Zpr1 is a bespoke chaperone essential for eEF1A biogenesis.
Capturing the conversion of the pathogenic alpha-1-antitrypsin fold by ATF6 enhanced proteostasis.
Modulation of protease expression by the transcription factor Ptx1/PITX regulates protein quality control during aging.
ERASing endoplasmic reticulum stress: the faster, the better.
Sec61 channel subunit Sbh1/Sec61β promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation.
Sensitive imaging of Endoplasmic reticulum (ER) autophagy with an acidity-reporting ER-Tracker.
The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER.
G3BP1-dependent mechanism suppressing protein aggregation in Huntington's models and its demise upon stress granule assembly.
Reduced sphingolipid biosynthesis modulates proteostasis networks to enhance longevity.
Ubiquitination of stalled ribosomes enables mRNA decay via HBS-1 and NONU-1 in vivo.
Copper-dependent autophagic degradation of GPX4 drives ferroptosis.
Assembly chaperone Nas6 selectively destabilizes 26S proteasomes with defective regulatory particle-core particle interfaces.
HSP70 mediates a crosstalk between the estrogen and the heat shock response pathways.
LYSET/TMEM251- a novel key component of the mannose-6-phosphate pathway.
Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression.
Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.
Electrical signals in the ER are cell type and stimulus specific with extreme spatial compartmentalization in neurons.
Nonhistone Lysine Methylation as a Protein Degradation Signal.
In-cell chemical crosslinking identifies hotspots for SQSTM-1/p62-IκBα interaction that underscore a critical role of p62 in limiting NF-κB activation through IκBα-stabilization.
Chaperone-mediated autophagy promotes PCa survival during ARPI through selective proteome remodeling.
RIPosomes are targets of IRGM-SQSTM1-dependent autophagy.
SPATA2 restricts OTULIN-dependent LUBAC activity independently of CYLD.
Perseverance of protein homeostasis despite mistranslation of glycine codons with alanine.
Preclinical mouse model of a misfolded PNLIP variant develops chronic pancreatitis.
EMC3 regulates mesenchymal cell survival via control of the mitotic spindle assembly.

Sci Adv. 2023 Jan 13. 9(2): eadd8579

The ERAD system is restricted by elevated ceramides.

Jiwon Hwang, Brian G Peterson, Jeffrey Knupp, Ryan D Baldridge.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as ER-associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, and ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition influences ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long-chain ceramides leads to a marked increase in the level of ubiquitinated substrates in the ER membrane and concomitantly reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized mechanism in which ER membrane lipid remodeling changes the activity of ERAD.

DOI: https://doi.org/10.1126/sciadv.add8579
Cell. 2023 Jan 05. pii: S0092-8674(22)01538-0. [Epub ahead of print]

An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes.

Keely Oltion, Jordan D Carelli, Tangpo Yang, Stephanie K See, Hao-Yuan Wang, Martin Kampmann, Jack Taunton.

  Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.

Keywords:  CRISPRi screen; E3 ligase; K6-linked ubiquitin; RWD domain; diGly proteomics; natural product; protein synthesis; ribosomal ubiquitination; ribosome quality control; translation factor

DOI:  https://doi.org/10.1016/j.cell.2022.12.025
Nat Commun. 2023 Jan 10. 14(1): 79

Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

Yoshitaka Matsuo, Takayuki Uchihashi, Toshifumi Inada.

The collision sensor Hel2 specifically recognizes colliding ribosomes and ubiquitinates the ribosomal protein uS10, leading to noncanonical subunit dissociation by the ribosome-associated quality control trigger (RQT) complex. Although uS10 ubiquitination is essential for rescuing stalled ribosomes, its function and recognition steps are not fully understood. Here, we show that the RQT complex components Cue3 and Rqt4 interact with the K63-linked ubiquitin chain and accelerate the recruitment of the RQT complex to the ubiquitinated colliding ribosome. The CUE domain of Cue3 and the N-terminal domain of Rqt4 bind independently to the K63-linked ubiquitin chain. Their deletion abolishes ribosomal dissociation mediated by the RQT complex. High-speed atomic force microscopy (HS-AFM) reveals that the intrinsically disordered regions of Rqt4 enable the expansion of the searchable area for interaction with the ubiquitin chain. These findings provide mechanistic insight into the decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

DOI: https://doi.org/10.1038/s41467-022-35608-4
Biochim Biophys Acta Gene Regul Mech. 2023 Jan 10. pii: S1874-9399(23)00003-2. [Epub ahead of print] 194908

The cellular pathways that maintain the quality control and transport of diverse potassium channels.

Nga H Nguyen, Jeffrey L Brodsky.

  Potassium channels are multi-subunit transmembrane proteins that permit the selective passage of potassium and play fundamental roles in physiological processes, such as action potentials in the nervous system and organismal salt and water homeostasis, which is mediated by the kidney. Like all ion channels, newly translated potassium channels enter the endoplasmic reticulum (ER) and undergo the error-prone process of acquiring post-translational modifications, folding into their native conformations, assembling with other subunits, and trafficking through the secretory pathway to reach their final destinations, most commonly the plasma membrane. Disruptions in these processes can result in detrimental consequences, including various human diseases. Thus, multiple quality control checkpoints evolved to guide potassium channels through the secretory pathway and clear potentially toxic, aggregation-prone misfolded species. We will summarize current knowledge on the mechanisms underlying potassium channel quality control in the secretory pathway, highlight diseases associated with channel misfolding, and suggest potential therapeutic routes.

Keywords:  Endocytosis; Endoplasmic reticulum associated degradation (ERAD); Endosomal sorting; Molecular chaperones; Plasma membrane quality control; Protein quality control

DOI:  https://doi.org/10.1016/j.bbagrm.2023.194908
Nat Commun. 2023 Jan 11. 14(1): 168

The unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family.

Xiangyi S Wang, Thomas R Cotton, Sarah J Trevelyan, Lachlan W Richardson, Wei Ting Lee, John Silke, Bernhard C Lechtenberg.

The RING-between-RING (RBR) E3 ubiquitin ligase family in humans comprises 14 members and is defined by a two-step catalytic mechanism in which ubiquitin is first transferred from an E2 ubiquitin-conjugating enzyme to the RBR active site and then to the substrate. To define the core features of this catalytic mechanism, we here structurally and biochemically characterise the two RBRs HOIL-1 and RNF216. Crystal structures of both enzymes in their RBR/E2-Ub/Ub transthiolation complexes capturing the first catalytic step, together with complementary functional experiments, reveal the defining features of the RBR catalytic mechanism. RBRs catalyse ubiquitination via a conserved transthiolation complex structure that enables efficient E2-to-RBR ubiquitin transfer. Our data also highlight a conserved RBR allosteric activation mechanism by distinct ubiquitin linkages that suggests RBRs employ a feed-forward mechanism. We finally identify that the HOIL-1 RING2 domain contains an unusual Zn2/Cys6 binuclear cluster that is required for catalytic activity and substrate ubiquitination.

DOI: https://doi.org/10.1038/s41467-023-35871-z
Cell Mol Life Sci. 2023 Jan 07. 80(1): 32

HSP70-binding motifs function as protein quality control degrons.

Amanda B Abildgaard, Vasileios Voutsinos, Søren D Petersen, Fia B Larsen, Caroline Kampmeyer, Kristoffer E Johansson, Amelie Stein, Tommer Ravid, Claes Andréasson, Michael K Jensen, Kresten Lindorff-Larsen, Rasmus Hartmann-Petersen.

  Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.

Keywords:  Chaperone; Proteasome; Protein degradation; Protein quality control; Protein stability; Protein unfolding

DOI:  https://doi.org/10.1007/s00018-022-04679-3
Mol Cell. 2023 Jan 04. pii: S1097-2765(22)01169-8. [Epub ahead of print]

Zinc-finger protein Zpr1 is a bespoke chaperone essential for eEF1A biogenesis.

Ibrahim M Sabbarini, Dvir Reif, Alexander J McQuown, Anjali R Nelliat, Jeffrey Prince, Britnie Santiago Membreno, Colin Chih-Chien Wu, Andrew W Murray, Vladimir Denic.

  The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to eEF1A insufficiency, thereby inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we show by using two distinct biochemical reconstitution approaches that Zpr1 enables eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism mediated by the Zpr1's zinc-finger and alpha-helical hairpin structures. Our work uncovers the long-sought-after function of Zpr1 as a bespoke chaperone tailored to the biogenesis of one of the most abundant proteins in the cell.

Keywords:  chaperone; heat shock response; integrated stress response; misfolding; translation elongation

DOI:  https://doi.org/10.1016/j.molcel.2022.12.012
Cell Chem Biol. 2022 Dec 31. pii: S2451-9456(22)00454-8. [Epub ahead of print]

Capturing the conversion of the pathogenic alpha-1-antitrypsin fold by ATF6 enhanced proteostasis.

Shuhong Sun, Chao Wang, Pei Zhao, Gabe M Kline, Julia M D Grandjean, Xin Jiang, Richard Labaudiniere, R Luke Wiseman, Jeffery W Kelly, William E Balch.

  Genetic variation in alpha-1 antitrypsin (AAT) causes AAT deficiency (AATD) through liver aggregation-associated gain-of-toxic pathology and/or insufficient AAT activity in the lung manifesting as chronic obstructive pulmonary disease (COPD). Here, we utilize 71 AATD-associated variants as input through Gaussian process (GP)-based machine learning to study the correction of AAT folding and function at a residue-by-residue level by pharmacological activation of the ATF6 arm of the unfolded protein response (UPR). We show that ATF6 activators increase AAT neutrophil elastase (NE) inhibitory activity, while reducing polymer accumulation for the majority of AATD variants, including the prominent Z variant. GP-based profiling of the residue-by-residue response to ATF6 activators captures an unexpected role of the "gate" area in managing AAT-specific activity. Our work establishes a new spatial covariant (SCV) understanding of the convertible state of the protein fold in response to genetic perturbation and active environmental management by proteostasis enhancement for precision medicine.

Keywords:  Gaussian process; activating transcription factor 6 (ATF6); alpha-1 antitrypsin; alpha-1 antitrypsin deficiency; chaperones; genetic variation; machine learning; pharmacological ATF6 activators; precision medicine; protein aggregation; protein folding; protein misfolding disease; proteostasis; unfolded protein response (UPR)

DOI:  https://doi.org/10.1016/j.chembiol.2022.12.004
Cell Rep. 2023 Jan 10. pii: S2211-1247(22)01874-5. [Epub ahead of print]42(1): 111970

Modulation of protease expression by the transcription factor Ptx1/PITX regulates protein quality control during aging.

Jianqin Jiao, Michelle Curley, Flavia A Graca, Maricela Robles-Murguia, Abbas Shirinifard, David Finkelstein, Beisi Xu, Yiping Fan, Fabio Demontis.

  Protein quality control is important for healthy aging and is dysregulated in age-related diseases. The autophagy-lysosome and ubiquitin-proteasome are key for proteostasis, but it remains largely unknown whether other proteolytic systems also contribute to maintain proteostasis during aging. Here, we find that expression of proteolytic enzymes (proteases/peptidases) distinct from the autophagy-lysosome and ubiquitin-proteasome systems declines during skeletal muscle aging in Drosophila. Age-dependent protease downregulation undermines proteostasis, as demonstrated by the increase in detergent-insoluble poly-ubiquitinated proteins and pathogenic huntingtin-polyQ levels in response to protease knockdown. Computational analyses identify the transcription factor Ptx1 (homologous to human PITX1/2/3) as a regulator of protease expression. Consistent with this model, Ptx1 protein levels increase with aging, and Ptx1 RNAi counteracts the age-associated downregulation of protease expression. Moreover, Ptx1 RNAi improves muscle protein quality control in a protease-dependent manner and extends lifespan. These findings indicate that proteases and their transcriptional modulator Ptx1 ensure proteostasis during aging.

Keywords:  CP: Cell biology; CP: Molecular biology; PITX; Ptx1; aging; betaTry; huntingtin; peptidase; protease; protein quality control; proteostasis; skeletal muscle

DOI:  https://doi.org/10.1016/j.celrep.2022.111970
Trends Cell Biol. 2023 Jan 07. pii: S0962-8924(22)00277-X. [Epub ahead of print]

ERASing endoplasmic reticulum stress: the faster, the better.

Jogender Singh.

  The endoplasmic reticulum (ER) has evolved multiple mechanisms to maintain homeostasis under stress conditions. A recent study by Efstathiou et al. identified a novel mechanism of silencing ER-associated RNAs by the exogenous RNA interference pathway. This adaptive response reduces protein flux in the ER under stressful conditions.

Keywords:  ERAD; RNA interference; RNA silencing; endoplasmic reticulum

DOI:  https://doi.org/10.1016/j.tcb.2022.12.003
J Biol Chem. 2023 Jan 10. pii: S0021-9258(23)00027-3. [Epub ahead of print] 102895

Sec61 channel subunit Sbh1/Sec61β promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation.

Guido Barbieri, Julien Simon, Cristina R Lupusella, Fabio Pereira, Francesco Elia, Hadar Meyer, Maya Schuldiner, Steven D Hanes, Duy Nguyen, Volkhard Helms, Karin Römisch.

  The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one non-essential subunit, Sec61β/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.

Keywords:  Ess1/PIN1; Sec61 channel; endoplasmic reticulum (ER); protein isomerase; protein phosphorylation; protein secretion; protein translocation

DOI:  https://doi.org/10.1016/j.jbc.2023.102895
Autophagy. 2023 Jan 12. 1-11

Sensitive imaging of Endoplasmic reticulum (ER) autophagy with an acidity-reporting ER-Tracker.

Yilong Shi, Xiaoxue Zou, Xianghui Zheng, Yimin Wu, Jiahuai Han, Shoufa Han.

  Macroautophagic/autophagic turnover of endoplasmic reticulum (reticulophagy) is critical for cell health. Herein we reported a sensitive fluorescence-on imaging of reticulophagy using a small molecule probe (ER-proRed) comprised of green-emissive fluorinated rhodol for ER targeting and nonfluorescent rhodamine-lactam prone to lysosome-triggered red fluorescence. Partitioned in ER to exhibit green fluorescence, ER-proRed gives intense red fluorescence upon co-delivery with ER into acidic lysosomes. Serving as the signal of reticulophagy, the turning on of red fluorescence enables discernment of reticulophagy induced by starvation, varied levels of reticulophagic receptors, and chemical agents such as etoposide and sodium butyrate. These results show ER probes optically activatable in lysosomes, such as ER-proRed, offer a sensitive and simplified tool for studying reticulophagy in biology and diseases.Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CQ, chloroquine diphosphate; ER, endoplasmic reticulum; FHR, fluorinated hydrophobic rhodol; GFP, green fluorescent protein; Reticulophagy, selective autophagy of ER; RFP, red fluorescent protein; ROX, X-rhodamine; UPR, unfolded protein response.

Keywords:  Autophagy imaging; endoplasmic reticulum; fluorescence-on; lysosomal acidity; reticulophagy

DOI:  https://doi.org/10.1080/15548627.2023.2165759
Cell Rep. 2022 Dec 28. pii: S2211-1247(22)01819-8. [Epub ahead of print]42(1): 111921

The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER.

Paul Heo, Jacob A Culver, Jennifer Miao, Frederic Pincet, Malaiyalam Mariappan.

  Tail-anchored (TA) proteins contain a single C-terminal transmembrane domain (TMD) that is captured by the cytosolic Get3 in yeast (TRC40 in humans). Get3 delivers TA proteins to the Get1/2 complex for insertion into the endoplasmic reticulum (ER) membrane. How Get1/2 mediates insertion of TMDs of TA proteins into the membrane is poorly understood. Using bulk fluorescence and microfluidics assays, we show that Get1/2 forms an aqueous channel in reconstituted bilayers. We estimate the channel diameter to be ∼2.5 nm wide, corresponding to the circumference of two Get1/2 complexes. We find that the Get3 binding can seal the Get1/2 channel, which dynamically opens and closes. Our mutation analysis further shows that the Get1/2 channel activity is required to release TA proteins from Get3 for insertion into the membrane. Hence, we propose that the Get1/2 channel functions as an insertase for insertion of TMDs and as a translocase for translocation of C-terminal hydrophilic segments.

Keywords:  CP: Cell biology; CP: Molecular biology; channel; membrane protein insertion; protein translocation; tail-anchored proteins

DOI:  https://doi.org/10.1016/j.celrep.2022.111921
Hum Mol Genet. 2023 Jan 05. pii: ddac304. [Epub ahead of print]

G3BP1-dependent mechanism suppressing protein aggregation in Huntington's models and its demise upon stress granule assembly.

Ricardo Gutiérrez-Garcia, Seda Koyuncu, Franziska Hommen, Saygın Bilican, Hyun Ju Lee, Azra Fatima, David Vilchez.

Stress granules are membrane-less ribonucleoprotein organelles that assemble upon exposure to stress conditions, but rapidly disassemble upon removal of stress. However, chronic stress can lead to persistent stress granules, a feature of distinct age-related neurodegenerative disorders. Among them, Huntington's disease (HD) which is caused by mutant expansion of the polyglutamine (polyQ) repeats of huntingtin protein (HTT), leading to its aggregation. To identify modulators of mutant HTT aggregation, we define its interactome in striatal neurons from patient-derived induced pluripotent stem cells (HD-iPSCs). We find that HTT interacts with G3BP1, a characteristic component of stress granules. Knockdown of G3BP1 increases mutant HTT protein levels and abolishes the ability of iPSCs as well as their differentiated neural counterparts to suppress mutant HTT aggregation. Moreover, loss of G3BP1 hastens polyQ-expanded aggregation and toxicity in the neurons of HD C. elegans models. Likewise, the assembly of G3BP1 into stress granules upon distinct stress conditions also reduces its interaction with HTT in human cells, promoting mutant HTT aggregation. Notably, enhancing the levels of G3BP1 is sufficient to induce proteasomal degradation of mutant HTT and prevent its aggregation, whereas the formation of stress granules blocks these ameliorative effects. In contrast, a mutant G3BP1 variant that cannot accumulate into granules retains its capacity to prevent mutant HTT aggregation even when the cells assemble stress granules. Thus, our findings indicate a direct role of G3BP1 and stress granule assembly in mutant HTT aggregation that may have implications for HD.

DOI: https://doi.org/10.1093/hmg/ddac304
Aging (Albany NY). 2023 Jan 14. 15

Reduced sphingolipid biosynthesis modulates proteostasis networks to enhance longevity.

Nathaniel L Hepowit, Eric Blalock, Sangderk Lee, Kimberly M Bretland, Jason A MacGurn, Robert C Dickson.

  As the elderly population increases, chronic, age-associated diseases are challenging healthcare systems around the world. Nutrient limitation is well known to slow the aging process and improve health. Regrettably, practicing nutrient restriction to improve health is unachievable for most people. Alternatively, pharmacological strategies are being pursued including myriocin which increases lifespan in budding yeast. Myriocin impairs sphingolipid synthesis, resulting in lowered amino acid pools which promote entry into a quiescent, long-lived state. Here we present transcriptomic data during the first 6 hours of drug treatment that improves our mechanistic understanding of the cellular response to myriocin and reveals a new role for ubiquitin in longevity. Previously we found that the methionine transporter Mup1 traffics to the plasma membrane normally in myriocin-treated cells but is not active and undergoes endocytic clearance. We now show that UBI4, a gene encoding stressed-induced ubiquitin, is vital for myriocin-enhanced lifespan. Furthermore, we show that Mup1 fused to a deubiquitinase domain impairs myriocin-enhanced longevity. Broader effects of myriocin treatment on ubiquitination are indicated by our finding of a significant increase in K63-linked ubiquitin polymers following myriocin treatment. Although proteostasis is broadly accepted as a pillar of aging, our finding that ubiquitination of an amino acid transporter promotes longevity in myriocin-treated cells is novel. Addressing the role of ubiquitination/deubiquitination in longevity has the potential to reveal new strategies and targets for promoting healthy aging.

Keywords:  amino acid transport; longevity; proteostasis; sphingolipid; ubiquitin

DOI:  https://doi.org/10.18632/aging.204485
PLoS Genet. 2023 Jan 10. 19(1): e1010577

Ubiquitination of stalled ribosomes enables mRNA decay via HBS-1 and NONU-1 in vivo.

Parissa C Monem, Nitin Vidyasagar, Audrey L Piatt, Enisha Sehgal, Joshua A Arribere.

As ribosomes translate the genetic code, they can encounter a variety of obstacles that hinder their progress. If ribosomes stall for prolonged times, cells suffer due to the loss of translating ribosomes and the accumulation of aberrant protein products. Thus to protect cells, stalled ribosomes experience a series of reactions to relieve the stall and degrade the offending mRNA, a process known as No-Go mRNA Decay (NGD). While much of the machinery for NGD is known, the precise ordering of events and factors along this pathway has not been tested. Here, we deploy C. elegans to unravel the coordinated events comprising NGD. Utilizing a novel reporter and forward and reverse genetics, we identify the machinery required for NGD. Our subsequent molecular analyses define a functional requirement for ubiquitination on at least two ribosomal proteins (eS10 and uS10), and we show that ribosomes lacking ubiquitination sites on eS10 and uS10 fail to perform NGD in vivo. We show that the nuclease NONU-1 acts after the ubiquitin ligase ZNF-598, and discover a novel requirement for the ribosome rescue factors HBS-1/PELO-1 in mRNA decay via NONU-1. Taken together, our work demonstrates mechanisms by which ribosomes signal to effectors of mRNA repression, and we delineate links between repressive factors working toward a well-defined NGD pathway.

DOI: https://doi.org/10.1371/journal.pgen.1010577
Autophagy. 2023 Jan 12. 1-15

Copper-dependent autophagic degradation of GPX4 drives ferroptosis.

Qian Xue, Ding Yan, Xi Chen, Xiaofen Li, Rui Kang, Daniel J Klionsky, Guido Kroemer, Xin Chen, Daolin Tang, Jinbao Liu.

  Ferroptosis is a type of iron-dependent regulated cell death characterized by unrestricted lipid peroxidation and membrane damage. Although GPX4 (glutathione peroxidase 4) plays a master role in blocking ferroptosis by eliminating phospholipid hydroperoxides, the regulation of GPX4 remains poorly understood. Here, we report an unexpected role for copper in promoting ferroptotic cell death, but not cuproptosis, by inducing macroautophagic/autophagic degradation of GPX4. Copper chelators reduce ferroptosis sensitivity but do not inhibit other types of cell death, such as apoptosis, necroptosis, and alkaliptosis. Conversely, exogenous copper increases GPX4 ubiquitination and the formation of GPX4 aggregates by directly binding to GPX4 protein cysteines C107 and C148. TAX1BP1 (Tax1 binding protein 1) then acts as an autophagic receptor for GPX4 degradation and subsequent ferroptosis in response to copper stress. Consequently, copper enhances ferroptosis-mediated tumor suppression in a mouse model of pancreatic cancer tumor, whereas copper chelators attenuate experimental acute pancreatitis associated with ferroptosis. Taken together, these findings provide new insights into the link between metal stress and autophagy-dependent cell death.Abbreviations: CALCOCO2, calcium binding and coiled-coil domain 2; GPX4, glutathione peroxidase 4; MAP1LC3A/B, microtubule associated protein 1 light chain 3 alpha/beta; MPO, myeloperoxidase; NCOA4, nuclear receptor coactivator 4; OPTN, optineurin; PDAC, pancreatic ductal adenocarcinoma; RIPK1, receptor interacting serine/threonine kinase 1; ROS, reactive oxygen species; SLC40A1, solute carrier family 40 member 1; SQSTM1, sequestosome 1; TAX1BP1, Tax1 binding protein 1; TEPA, tetraethylenepentamine; TM, tetrathiomolybdate.

Keywords:  Autophagy; GPX4; TAX1BP1; copper; cuproptosis; ferroptosis

DOI:  https://doi.org/10.1080/15548627.2023.2165323
J Biol Chem. 2023 Jan 09. pii: S0021-9258(23)00026-1. [Epub ahead of print] 102894

Assembly chaperone Nas6 selectively destabilizes 26S proteasomes with defective regulatory particle-core particle interfaces.

Jennifer L Warnock, Gabriel W Jobin, Sandhya Kumar, Robert J Tomko.

The 26S proteasome is a 66-subunit chambered protease present in all eukaryotes that maintains organismal health by degrading unneeded or defective proteins. Defects in proteasome function or assembly are known to contribute to the development of various cancers, neurodegeneration, and diabetes. During proteasome biogenesis, a family of evolutionarily conserved chaperones assemble a hexameric ring of AAA+ family ATPase subunits contained within the proteasomal regulatory particle (RP) and guide their docking onto the surface of the proteolytic core particle (CP). This RP-CP interaction couples the substrate capture and unfolding process to proteolysis. We previously reported a mutation in the proteasome that promoted dissociation of the RP and CP by one of these chaperones, Nas6. However, the nature of the signal for Nas6-dependent proteasome disassembly, and the generality of this post-assembly proteasome quality control function for Nas6 remains unknown. Here, we use structure-guided mutagenesis and in vitro proteasome disassembly assays to demonstrate that Nas6 more broadly destabilizes 26S proteasomes with a defective RP-CP interface. We show that Nas6 can promote dissociation of mature proteasomes into RP and CP in cells harboring defects on either side of the RP-CP interface. This function is unique to Nas6 and independent from other known RP assembly chaperones. Further biochemical experiments suggest that Nas6 may exploit a weakened RP-CP interface to dissociate the RP from the CP. We propose that this post-assembly role of Nas6 may fulfill a quality control function in cells by promoting the recycling of functional subcomplexes contained within defective proteasomes.

DOI: https://doi.org/10.1016/j.jbc.2023.102894
J Biol Chem. 2023 Jan 04. pii: S0021-9258(23)00004-2. [Epub ahead of print] 102872

HSP70 mediates a crosstalk between the estrogen and the heat shock response pathways.

Maruhen Ad Silveira, Fatemeh Khadangi, Sofiane Yacine Mersaoui, Jean-Yves Masson, Divya Naik, Steve Bilodeau.

  Cells respond to multiple signals from the environment simultaneously which often creates crosstalk between pathways affecting the capacity to adapt to the changing environment. Chaperones are an important component in the cellular integration of multiple responses to environmental signals, often implicated in negative feedback and inactivation mechanisms. These mechanisms include the stabilization of steroid hormone nuclear receptors in the cytoplasm in the absence of their ligand. Here we show using immunofluorescence, chromatin immunoprecipitation and nascent transcripts production that the HSP70 chaperone plays a central role in a new crosstalk mechanism between the steroid and heat shock response pathways. HSP70-dependent feedback mechanisms are required to inactivate the heat shock factor 1 (HSF1) after activation. Interestingly, a steroid stimulation leads to faster accumulation of HSF1 in inactive foci following heat shock. Our results further show that in the presence of estrogen, HSP70 accumulates at HSF1-regulated noncoding regions, leading to deactivation of HSF1 and the abrogation of the heat shock transcriptional response. Using an HSP70 inhibitor, we demonstrate that the crosstalk between both pathways is dependent on the chaperone activity. These results suggest that HSP70 availability is a key determinant in the transcriptional integration of multiple external signals. Overall, these results offer a better understanding of the crosstalk between the heat shock and steroid responses, which are salient in neurodegenerative disorders and cancers.

Keywords:  HSP70 inhibitor; chaperones; feedback mechanism; steroids; transcription

DOI:  https://doi.org/10.1016/j.jbc.2023.102872
Autophagy. 2023 Jan 12.

LYSET/TMEM251- a novel key component of the mannose-6-phosphate pathway.

Wenjie Qiao, Christopher M Richards, Sabrina Jabs.

  Degradation of macromolecules delivered to lysosomes by processes such as autophagy or endocytosis is crucial for cellular function. Lysosomes require more than 60 soluble hydrolases in order to catabolize such macromolecules. These soluble hydrolases are tagged with mannose-6-phosphate (M6P) moieties in sequential reactions by the Golgi-resident GlcNAc-1-phosphotransferase complex and NAGPA/UCE/uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase), which allows their delivery to endosomal/lysosomal compartments through trafficking mediated by cation-dependent and -independent mannose-6-phosphate receptors (MPRs). We and others recently identified TMEM251 as a novel regulator of the M6P pathway via independent genome-wide genetic screening strategies. We renamed TMEM251 to LYSET (lysosomal enzyme trafficking factor) to establish nomenclature reflective to this gene's function. LYSET is a Golgi-localized transmembrane protein important for the retention of the GlcNAc-1-phosphotransferase complex in the Golgi-apparatus. The current understanding of LYSET's importance regarding human biology is 3-fold: 1) highly pathogenic viruses that depend on lysosomal hydrolase activity require LYSET for infection. 2) The presence of LYSET is critical for cancer cell proliferation in nutrient-deprived environments in which extracellular proteins must be catabolized. 3) Inherited pathogenic alleles of LYSET can cause a severe inherited disease which resembles GlcNAc-1-phosphotransferase deficiency (i.e., mucolipidosis type II).

Keywords:  GlcNAc-1-phosphotransferase; Golgi-apparatus; lysosomal enzyme trafficking; lysosome; mannose-6-phosphate; mucolipidosis type II

DOI:  https://doi.org/10.1080/15548627.2023.2167376
J Cell Biol. 2023 Mar 06. pii: e202206058. [Epub ahead of print]222(3):

Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression.

Helen M Vander Wende, Mounika Gopi, Megan Onyundo, Claudia Medrano, Temiloluwa Adanlawo, Gloria Ann Brar.

Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case-Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients-is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1's role in human disease.

DOI: https://doi.org/10.1083/jcb.202206058
Blood Cancer J. 2023 Jan 12. 13(1): 12

Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.

Keiji Kurata, Anna-James Bott, Mark A Tye, Leona Yamamoto, Mehmet K Samur, Yu-Tzu Tai, James Dunford, Catrine Johansson, Filiz Senbabaoglu, Martin Philpott, Charlotte Palmer, Karthik Ramasamy, Sarah Gooding, Mihaela Smilova, Giorgia Gaeta, Manman Guo, John C Christianson, N Connor Payne, Kritika Singh, Kubra Karagoz, Matthew E Stokes, Maria Ortiz, Patrick Hagner, Anjan Thakurta, Adam Cribbs, Ralph Mazitschek, Teru Hideshima, Kenneth C Anderson, Udo Oppermann.

Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

DOI: https://doi.org/10.1038/s41408-023-00787-w
Cell Rep. 2023 Jan 05. pii: S2211-1247(22)01844-7. [Epub ahead of print]42(1): 111943

Electrical signals in the ER are cell type and stimulus specific with extreme spatial compartmentalization in neurons.

Evan P Campbell, Ahmed A Abushawish, Lauren A Valdez, Miriam K Bell, Melita Haryono, Padmini Rangamani, Brenda L Bloodgood.

  The endoplasmic reticulum (ER) is a tortuous organelle that spans throughout a cell with a continuous membrane containing ion channels, pumps, and transporters. It is unclear if stimuli that gate ER ion channels trigger substantial membrane potential fluctuations and if those fluctuations spread beyond their site of origin. Here, we visualize ER membrane potential dynamics in HEK cells and cultured rat hippocampal neurons by targeting a genetically encoded voltage indicator specifically to the ER membrane. We report the existence of clear cell-type- and stimulus-specific ER membrane potential fluctuations. In neurons, direct stimulation of ER ryanodine receptors generates depolarizations that scale linearly with stimulus strength and reach tens of millivolts. However, ER potentials do not spread beyond the site of receptor activation, exhibiting steep attenuation that is exacerbated by intracellular large conductance K+ channels. Thus, segments of ER can generate large depolarizations that are actively restricted from impacting nearby, contiguous membrane.

Keywords:  BK channels; CP: Cell biology; CP: Neuroscience; ER; HEK cells; dendrites; electrical compartmentalization; genetically encoded voltage sensor; membrane potential; neurons

DOI:  https://doi.org/10.1016/j.celrep.2022.111943
J Chem. 2022 ;pii: 1969299. [Epub ahead of print]2022

Nonhistone Lysine Methylation as a Protein Degradation Signal.

Nicholas A Lehning, Brad E Morrison.

Protein degradation is a fundamental feature of cellular life, and malfunction of this process is implicated in human disease. Ubiquitin tagging is the best characterized mechanism of targeting a protein for degradation; however, there are a growing number of distinct mechanisms which have also been identified that carry out this essential function. For example, covalent tagging of proteins with sequestosome-1 targets them for selective autophagy. Degradation signals are not exclusively polypeptides such as ubiquitin, NEDD8, and sequestosome-1. Phosphorylation, acetylation, and methylation are small covalent additions that can also direct protein degradation. The diversity of substrate sequences and overlap with other pleotrophic functions for these smaller signaling moieties has made their characterization more challenging. However, these small signals might be responsible for orchestrating a large portion of the protein degradation activity in the cell. As such, there has been increasing interest in lysine methylation and associated lysine methyltransferases (KMTs), beyond canonical histone protein modification, in mediating protein degradation in a variety of contexts. This review focuses on the current evidence for lysine methylation as a protein degradation signal with a detailed discussion of the class of enzymes responsible for this phenomenon.

DOI: https://doi.org/10.1155/2022/1969299
Mol Cell Proteomics. 2023 Jan 09. pii: S1535-9476(23)00004-X. [Epub ahead of print] 100495

In-cell chemical crosslinking identifies hotspots for SQSTM-1/p62-IκBα interaction that underscore a critical role of p62 in limiting NF-κB activation through IκBα-stabilization.

Yi Liu, Michael J Trnka, Liang He, A L Burlingame, Maria Almira Correia.

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with IκBα, an inhibitor of the transcriptional activator NF-κB. Such an intimate p62-IκBα association we now document leads to a marked 18-fold proteolytic IκBα-stabilization, enabling its nuclear entry and termination of the NF-κB-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-IκBα association. Accordingly, we have identified such IκBα hotspots to reside around N-terminal (K38, K47 and K67) and C-terminal (K238/C239) residues in its 5th ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) in the intervening region between its ZZ- and TB-subdomains. APEX proximity analyses upon IκBα co-transfection of cells with and without p62 have enabled the characterization of the p62 influence on IκBα-protein-protein interactions. Interestingly, consistent with p62's capacity to proteolytically stabilize IκBα, its presence greatly impaired IκBα's interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62-interaction with IκBα on an interface opposite to that of its NF-κB-interacting interface, p62 failed to significantly affect IκBα-NF-κB interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling, leads us to speculate that it may be involved in "piggy-back" nuclear transport of IκBα following its NF-κB-elicited transcriptional activation and de novo synthesis, required for termination of the NF-κB-activation cycle. Consequently, mice carrying a liver specific deletion of p62-residues 68-252, reveal age-dependent enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-κB.

DOI: https://doi.org/10.1016/j.mcpro.2023.100495
Oncogene. 2023 Jan 07.

Chaperone-mediated autophagy promotes PCa survival during ARPI through selective proteome remodeling.

Nicholas Nikesitch, Eliana Beraldi, Fan Zhang, Hans Adomat, Robert Bell, Kotaro Suzuki, Ladan Fazli, Sonia Hy Kung, Christopher Wells, Nicholas Pinette, Neetu Saxena, Yuzhuo Wang, Martin Gleave.

The androgen receptor (AR) plays an important role in PCa metabolism, with androgen receptor pathway inhibition (ARPI) subjecting PCa cells to acute metabolic stress caused by reduced biosynthesis and energy production. Defining acute stress response mechanisms that alleviate ARPI stress and therefore mediate prostate cancer (PCa) treatment resistance will help improve therapeutic outcomes of patients treated with ARPI. We identified the up-regulation of chaperone-mediated autophagy (CMA) in response to acute ARPI stress, which persisted in castration-resistant PCa (CRPC); previously undefined in PCa. CMA is a selective protein degradation pathway and a key stress response mechanism up-regulated under several stress stimuli, including metabolic stress. Through selective protein degradation, CMA orchestrates the cellular stress response by regulating cellular pathways through selective proteome remodeling. Through broad-spectrum proteomic analysis, CMA coordinates metabolic reprogramming of PCa cells to sustain PCa growth and survival during ARPI; through the upregulation of mTORC1 signaling and pathways associated with PCa biosynthesis and energetics. This not only promoted PCa growth during ARPI, but also promoted the emergence of CRPC in-vivo. During CMA inhibition, PCa metabolism is compromised, leading to ATP depletion, resulting in a profound anti-proliferative effect on PCa cells, and is enhanced when combined with ARPI. Furthermore, CMA inhibition prevented in-vivo tumour formation, and also re-sensitized enzalutamide-resistant cell lines in-vitro. The profound anti-proliferative effect of CMA inhibition was attributed to cell cycle arrest mediated through p53 transcriptional repression of E2F target genes. In summary, CMA is an acute ARPI stress response mechanism, essential in alleviating ARPI induced metabolic stress, essential for ensuring PCa growth and survival. CMA plays a critical role in the development of ARPI resistance in PCa.

DOI: https://doi.org/10.1038/s41388-022-02573-7
Autophagy. 2023 Jan 10.

RIPosomes are targets of IRGM-SQSTM1-dependent autophagy.

Subhash Mehto, Soumya Kundu, Swati Chauhan, Santosh Chauhan.

  The NOD1-NOD2-RIPK2-NFKB/NF-κB pro-inflammatory axis plays a significant role in regulating the immune response to bacterial infection. However, an excess of NFKB-dependent cytokine response can be detrimental and, thus, should be kept under control to maintain the innate immune balance. In our recent study, first, we showed that bacterial infection induces the biogenesis of RIPK2 oligomers (RIPosomes) that are recruited around the bacteria to enhance an NFKB-dependent pro-inflammatory response. Next, we showed that SQSTM1- and IRGM-dependent selective macroautophagy/autophagy degrades RIPosomes and their components to limit NOD1-NOD2-RIPK2-NFKB pro-inflammatory signaling. Consistently, depletion of IRGM results in an augmented RIPK2-dependent pro-inflammatory cytokine response induced by Shigella flexneri and Salmonella typhimurium. Further, bacterial infection- and DSS-induced gut inflammation in irgm1KO mice is dampened upon therapeutic inhibition of RIPK2. Taken together, we showed that autophagy selectively degrades RIPosomes to suppress inflammation and maintain innate immune homeostasis.

Keywords:  Autophagy; IRGM; NFKB; NOD1; NOD2; RIPK2; RIPosomes

DOI:  https://doi.org/10.1080/15548627.2023.2166724
Cell Rep. 2023 Jan 12. pii: S2211-1247(22)01865-4. [Epub ahead of print]42(1): 111961

SPATA2 restricts OTULIN-dependent LUBAC activity independently of CYLD.

Laura Griewahn, Madeleine Müller, Lukas Peintner, Manuela Wissler, Martina Weiss, Prisca Brauns-Schubert, Ramin Massoumi, Christoph Borner, Olaf Groß, Monica Yabal, Céline Charvet, Ulrich Maurer.

  SPATA2 mediates the recruitment of CYLD to immune receptor complexes by bridging the interaction of CYLD with the linear ubiquitylation assembly complex (LUBAC) component HOIP. Whether SPATA2 exhibits functions independently of CYLD is unclear. Here, we show that, while Cyld-/- and Spata2-/- mice are viable, double mutants exhibit highly penetrant perinatal lethality, indicating independent functions of SPATA2 and CYLD. Cyld-/-Spata2-/- fibroblasts show increased M1-linked TNFR1-SC ubiquitylation and, similar to Cyld-/-Spata2-/- macrophages and intestinal epithelial cells, elevated pro-inflammatory gene expression compared with Cyld-/- or Spata2-/- cells. We show that SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and its zinc finger domain, thereby promoting autoubiquitylation of LUBAC. Consistently, increased pro-inflammatory signaling in Cyld-/-Spata2-/- cells depends on the presence of OTULIN. Our data therefore indicate that SPATA2 counteracts, independently of CYLD, the deubiquitylation of LUBAC by OTULIN and thereby attenuates LUBAC activity and pro-inflammatory signaling.

Keywords:  CP: Immunology; CP: Molecular biology; CYLD; LUBAC; OTULIN; SPATA2; apoptosis; cell death; inflammation; ubiquitylation

DOI:  https://doi.org/10.1016/j.celrep.2022.111961
Philos Trans R Soc Lond B Biol Sci. 2023 Feb 27. 378(1871): 20220029

Perseverance of protein homeostasis despite mistranslation of glycine codons with alanine.

Farah Hasan, Jeremy T Lant, Patrick O'Donoghue.

  By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNASer variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Keywords:  anticodon; genetic code ambiguity; identity element; mistranslation; protein quality control; transfer RNA

DOI:  https://doi.org/10.1098/rstb.2022.0029
Gut. 2023 Jan 11. pii: gutjnl-2022-327960. [Epub ahead of print]

Preclinical mouse model of a misfolded PNLIP variant develops chronic pancreatitis.

Guoying Zhu, Steven J Wilhelm, Leah G George, Brett M Cassidy, Sammy Zino, Cliff J Luke, Mina Hanna, Stephen Stone, Nhung Phan, Neel Matiwala, Samuel J Ballentine, Mark E Lowe, Xunjun Xiao.

   OBJECTIVE: Increasing evidence implicates mutation-induced protein misfolding and endoplasm reticulum (ER) stress in the pathophysiology of chronic pancreatitis (CP). The paucity of animal models harbouring genetic risk variants has hampered our understanding of how misfolded proteins trigger CP. We previously showed that pancreatic triglyceride lipase (PNLIP) p.T221M, a variant associated with steatorrhoea and possibly CP in humans, misfolds and elicits ER stress in vitro suggesting proteotoxicity as a potential disease mechanism. Our objective was to create a mouse model to determine if PNLIP p.T221M causes CP and to define the mechanism.
DESIGN: We created a mouse model of Pnlip p.T221M and characterised the structural and biochemical changes in the pancreas aged 1-12 months. We used multiple methods including histochemistry, immunostaining, transmission electron microscopy, biochemical assays, immunoblotting and qPCR.
RESULTS: We demonstrated the hallmarks of human CP in Pnlip p.T221M homozygous mice including progressive pancreatic atrophy, acinar cell loss, fibrosis, fatty change, immune cell infiltration and reduced exocrine function. Heterozygotes also developed CP although at a slower rate. Immunoblot showed that pancreatic PNLIP T221M misfolded as insoluble aggregates. The level of aggregates in homozygotes declined with age and was much lower in heterozygotes at all ages. The Pnlip p.T221M pancreas had increased ER stress evidenced by dilated ER, increased Hspa5 (BiP) mRNA abundance and a maladaptive unfolded protein response leading to upregulation of Ddit3 (CHOP), nuclear factor-κB and cell death.
CONCLUSION: Expression of PNLIP p.T221M in a preclinical mouse model results in CP caused by ER stress and proteotoxicity of misfolded mutant PNLIP.

Keywords:  CELL DEATH; CHRONIC PANCREATITIS; PANCREATITIS

DOI:  https://doi.org/10.1136/gutjnl-2022-327960
iScience. 2023 Jan 20. 26(1): 105667

EMC3 regulates mesenchymal cell survival via control of the mitotic spindle assembly.

Xiaofang Tang, Wei Wei, John M Snowball, Ernesto S Nakayasu, Sheila M Bell, Charles Ansong, Xinhua Lin, Jeffrey A Whitsett.

  Eukaryotic cells transit through the cell cycle to produce two daughter cells. Dysregulation of the cell cycle leads to cell death or tumorigenesis. Herein, we found a subunit of the ER membrane complex, EMC3, as a key regulator of cell cycle. Conditional deletion of Emc3 in mouse embryonic mesoderm led to reduced size and patterning defects of multiple organs. Emc3 deficiency impaired cell proliferation, causing spindle assembly defects, chromosome mis-segregation, cell cycle arrest at G2/M, and apoptosis. Upon entry into mitosis, mesenchymal cells upregulate EMC3 protein levels and localize EMC3 to the mitotic centrosomes. Further analysis indicated that EMC3 works together with VCP to tightly regulate the levels and activity of Aurora A, an essential factor for centrosome function and mitotic spindle assembly: while overexpression of EMC3 or VCP degraded Aurora A, their loss led to increased Aurora A stability but reduced Aurora A phosphorylation in mitosis.

Keywords:  Biological sciences; Cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2022.105667