bims-proteo 2024-04-21 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024‒04‒21
thirty-one papers selected by
Eric Chevet, INSERM

Inability to rescue stalled ribosomes results in overactivation of the integrated stress response.
Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2.
Expanding the ligand spaces for E3 ligases for the design of protein degraders.
HOIL1 mediates MDA5 activation through ubiquitination of LGP2.
Attenuating ribosome load improves protein output from mRNA by limiting translation-dependent mRNA decay.
Tracing genetic diversity captures the molecular basis of misfolding disease.
Proteasome hyperactivation rewires the proteome enhancing stress resistance, proteostasis, lipid metabolism and ERAD in C. elegans.
Unraveling the landscapes and regulation of scanning, leaky scanning, and 48S initiation complex conformations.
Abundance of the Membrane Proteome in Yeast Cells Lacking Spc1, a Non-catalytic Subunit of the Signal Peptidase Complex.
Tertiary structure and conformational dynamics of the anti-amyloidogenic chaperone DNAJB6b at atomistic resolution.
ER stress signaling at the interphase between MASH and hepatocellular carcinoma.
Selective autophagy of macromolecular complexes: what does it take to be taken?
Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.
Type I interferon regulation by USP18 is a key vulnerability in cancer.
IRE1 signaling increases PERK expression during chronic ER stress.
UPF1 regulates mRNA stability by sensing poorly translated coding sequences.
Endoplasmic reticulum - condensate interactions in protein synthesis and secretion.
Structure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex.
A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation.
Lysosomal Degradation Targets Mutant Calreticulin and the Thrombopoietin Receptor in Myeloproliferative Neoplasms.
Molecular basis for pH sensing in the KDEL trafficking receptor.
Molecular characterization of Rft1, a membrane protein associated with congenital disorder of glycosylation type 1N.
Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells.
Orchestrating neuronal activity-dependent translation via the integrated stress response protein GADD34.
Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.
Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies.
The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis.
Viral infection and spread are inhibited by the polyubiquitination and downregulation of TRPV2 channel by the interferon-stimulated gene TRIM21.
Nuclear RNA homeostasis promotes systems-level coordination of cell fate and senescence.
tRNA modification reprogramming contributes to artemisinin resistance in Plasmodium falciparum.
Microglia-derived extracellular vesicles trigger age-related neurodegeneration upon DNA damage.

J Biol Chem. 2024 Apr 16. pii: S0021-9258(24)01791-5. [Epub ahead of print] 107290

Inability to rescue stalled ribosomes results in overactivation of the integrated stress response.

Ankanahalli N Nanjaraj Urs, Victor Lasehinde, Lucas Kim, Elesa McDonald, Liewei L Yan, Hani S Zaher.

  Endogenous and exogenous chemical agents are known to compromise the integrity of RNA and cause ribosome stalling and collisions. Recent studies have shown that collided ribosomes serve as sensors for multiple processes, including ribosome quality control (RQC) and the integrated stress response (ISR). Since RQC and the ISR have distinct downstream consequences, it is of great importance that organisms activate the appropriate process. We previously showed that RQC is robustly activated in response to collisions and suppresses the ISR activation. However, the molecular mechanics behind this apparent competition were not immediately clear. Here we show that Hel2 does not physically compete with factors of the ISR, but instead its ribosomal-protein ubiquitination activity, and downstream resolution of collided ribosomes, is responsible for suppressing the ISR. Introducing a mutation in the RING domain of Hel2 - which inhibits its ubiquitination activity and downstream RQC but imparts higher affinity of the factor for collided ribosomes - resulted in increased activation of the ISR upon MMS-induced alkylation stress. Similarly, mutating Hel2's lysine targets in uS10, which is responsible for RQC activation, resulted in increased Gcn4 target induction. Remarkably, the entire process of RQC appears to be limited by the action of Hel2, as the overexpression of this one factor dramatically suppressed activation of the ISR. Collectively, our data suggest that cells evolved Hel2 to bind collided ribosomes with a relatively high affinity, but kept its concentration relatively low, ensuring that it gets exhausted under stress conditions that cannot be resolved by quality control processes.

Keywords:  E3 ubiquitin ligase; Gcn2; Gcn4; Hel2; integrated stress response; mRNA decay; protein synthesis; ribosome; ribosome quality control; stalling

DOI:  https://doi.org/10.1016/j.jbc.2024.107290
Nat Commun. 2024 Apr 13. 15(1): 3215

Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2.

Arun Kumar, Veena Mathew, Peter C Stirling.

Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.

DOI: https://doi.org/10.1038/s41467-024-47615-8
Bioorg Med Chem. 2024 Apr 12. pii: S0968-0896(24)00132-9. [Epub ahead of print]105 117718

Expanding the ligand spaces for E3 ligases for the design of protein degraders.

Rahman Shah Zaib Saleem, Martin P Schwalm, Stefan Knapp.

  Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.

Keywords:  CRBN; Degrader design; Molecular glue; New E3-ligase ligands; PROTAC; VHL

DOI:  https://doi.org/10.1016/j.bmc.2024.117718
bioRxiv. 2024 Apr 03. pii: 2024.04.02.587772. [Epub ahead of print]

HOIL1 mediates MDA5 activation through ubiquitination of LGP2.

Deion Cheng, Junji Zhu, GuanQun Liu, Michaela U Gack, Donna A MacDuff.

The RIG-I-like receptors (RLRs), RIG-I and MDA5, are innate sensors of RNA virus infections that are critical for mounting a robust antiviral immune response. We have shown previously that HOIL1, a component of the Linear Ubiquitin Chain Assembly Complex (LUBAC), is essential for interferon (IFN) induction in response to viruses sensed by MDA5, but not for viruses sensed by RIG-I. LUBAC contains two unusual E3 ubiquitin ligases, HOIL1 and HOIP. HOIP generates methionine-1-linked polyubiquitin chains, whereas HOIL1 has recently been shown to conjugate ubiquitin onto serine and threonine residues. Here, we examined the differential requirement for HOIL1 and HOIP E3 ligase activities in RLR-mediated IFN induction. We determined that HOIL1 E3 ligase activity was critical for MDA5-dependent IFN induction, while HOIP E3 ligase activity played only a modest role in promoting IFN induction. HOIL1 E3 ligase promoted MDA5 oligomerization, its translocation to mitochondrial-associated membranes, and the formation of MAVS aggregates. We identified that HOIL1 can interact with and facilitate the ubiquitination of LGP2, a positive regulator of MDA5 oligomerization. In summary, our work identifies LGP2 ubiquitination by HOIL1 in facilitating the activation of MDA5 and the induction of a robust IFN response.

DOI: https://doi.org/10.1101/2024.04.02.587772
Cell Rep. 2024 Apr 15. pii: S2211-1247(24)00426-1. [Epub ahead of print]43(4): 114098

Attenuating ribosome load improves protein output from mRNA by limiting translation-dependent mRNA decay.

Alicia A Bicknell, David W Reid, Marissa C Licata, Adriana K Jones, Yi Min Cheng, Mengying Li, Chiaowen Joyce Hsiao, Christopher S Pepin, Mihir Metkar, Yevgen Levdansky, Brian R Fritz, Elizaveta A Andrianova, Ruchi Jain, Eugene Valkov, Caroline Köhrer, Melissa J Moore.

  Developing an effective mRNA therapeutic often requires maximizing protein output per delivered mRNA molecule. We previously found that coding sequence (CDS) design can substantially affect protein output, with mRNA variants containing more optimal codons and higher secondary structure yielding the highest protein outputs due to their slow rates of mRNA decay. Here, we demonstrate that CDS-dependent differences in translation initiation and elongation rates lead to differences in translation- and deadenylation-dependent mRNA decay rates, thus explaining the effect of CDS on mRNA half-life. Surprisingly, the most stable and highest-expressing mRNAs in our test set have modest initiation/elongation rates and ribosome loads, leading to minimal translation-dependent mRNA decay. These findings are of potential interest for optimization of protein output from therapeutic mRNAs, which may be achieved by attenuating rather than maximizing ribosome load.

Keywords:  CP: Molecular biology; mRNA decay; mRNA therapeutics; ribosome; translation

DOI:  https://doi.org/10.1016/j.celrep.2024.114098
Nat Commun. 2024 Apr 18. 15(1): 3333

Tracing genetic diversity captures the molecular basis of misfolding disease.

Pei Zhao, Chao Wang, Shuhong Sun, Xi Wang, William E Balch.

Genetic variation in human populations can result in the misfolding and aggregation of proteins, giving rise to systemic and neurodegenerative diseases that require management by proteostasis. Here, we define the role of GRP94, the endoplasmic reticulum Hsp90 chaperone paralog, in managing alpha-1-antitrypsin deficiency on a residue-by-residue basis using Gaussian process regression-based machine learning to profile the spatial covariance relationships that dictate protein folding arising from sequence variants in the population. Covariance analysis suggests a role for the ATPase activity of GRP94 in controlling the N- to C-terminal cooperative folding of alpha-1-antitrypsin responsible for the correction of liver aggregation and lung-disease phenotypes of alpha-1-antitrypsin deficiency. Gaussian process-based spatial covariance profiling provides a standard model built on covariant principles to evaluate the role of proteostasis components in guiding information flow from genome to proteome in response to genetic variation, potentially allowing us to intervene in the onset and progression of complex multi-system human diseases.

DOI: https://doi.org/10.1038/s41467-024-47520-0
bioRxiv. 2024 Apr 06. pii: 2024.04.04.588128. [Epub ahead of print]

Proteasome hyperactivation rewires the proteome enhancing stress resistance, proteostasis, lipid metabolism and ERAD in C. elegans.

David Salcedo-Tacuma, Nadeeem Asad, Giovanni Howells, Raymond Anderson, David M Smith.

Proteasome dysfunction is implicated in the pathogenesis of neurodegenerative diseases and age-related proteinopathies. Using a C. elegans model, we demonstrate that 20S proteasome hyperactivation, facilitated by 20S gate-opening, accelerates the targeting of intrinsically disordered proteins. This leads to increased protein synthesis, extensive rewiring of the proteome and transcriptome, enhanced oxidative stress defense, accelerated lipid metabolism, and peroxisome proliferation. It also promotes ER-associated degradation (ERAD) of aggregation-prone proteins, such as alpha-1 antitrypsin (ATZ) and various lipoproteins. Notably, our results reveal that 20S proteasome hyperactivation suggests a novel role in ERAD with broad implications for proteostasis-related disorders, simultaneously affecting lipid homeostasis and peroxisome proliferation. Furthermore, the enhanced cellular capacity to mitigate proteostasis challenges, alongside unanticipated acceleration of lipid metabolism is expected to contribute to the longevity phenotype of this mutant. Remarkably, the mechanism of longevity induced by 20S gate opening appears unique, independent of known longevity and stress-resistance pathways. These results support the therapeutic potential of 20S proteasome activation in mitigating proteostasis-related disorders broadly and provide new insights into the complex interplay between proteasome activity, cellular health, and aging.

DOI: https://doi.org/10.1101/2024.04.04.588128
Cell Rep. 2024 Apr 16. pii: S2211-1247(24)00454-6. [Epub ahead of print]43(5): 114126

Unraveling the landscapes and regulation of scanning, leaky scanning, and 48S initiation complex conformations.

Benjamin Weiss, Rivka Dikstein.

  Scanning and initiation are critical steps in translation. Here, we utilized translation complex profiling (TCP-seq) to investigate 48S organization and eIF4G1-eIF1 inhibition impact. We provide global views of scanning and leaky scanning, uncovering a central role of eIF4G1-eIF1 in their regulation. We confirm AUG context importance, with non-leaky genes featuring a Kozak context and cytosine at positions -1 and +5. Capturing 48S complexes associated with eIF1, eIF4G1, eIF3, and eIF2 through selective TCP-seq revealed that the eIF3-scanning ribosome is highly vulnerable to eIF4G1-eIF1 inhibition, and eIF1 tends to dissociate upon AUG recognition. Initiation-site footprint analysis revealed a class spanning -12 to +18/19 from the AUG, representing the entire 48S and enriched with eIF2, eIF1, and eIF4G1, indicative of early initiation. Another eIF3-dependent class extends up to +26 and exhibits reduced eIF2 and eIF4G1 association, suggesting a late/alternative initiation complex. Our analysis provides an overview of scanning, initiation, and evidence for conformational rearrangements in vivo.

Keywords:  48S; CP: Molecular biology; TCP-seq; eIF1; eIF2; eIF3; eIF4G1; i14G1-12; leaky scanning; small ribosomal subunit; translation initiation

DOI:  https://doi.org/10.1016/j.celrep.2024.114126
J Membr Biol. 2024 Apr 17.

Abundance of the Membrane Proteome in Yeast Cells Lacking Spc1, a Non-catalytic Subunit of the Signal Peptidase Complex.

Chewon Yim, Yeonji Chung, Sungjoon Son, Jeesoo Kim, Jong-Seo Kim, Hyun Kim.

  The signal peptidase complex (SPC) mediates processing of signal peptides of secretory precursors. But, recent studies show that the eukaryotic SPC also cleaves internal transmembrane segments of some membrane proteins, and its non-catalytic subunit, Spc1/SPCS1 plays a critical role in this process. To assess the impact of Spc1 on membrane proteostasis, we carried out quantitative proteomics of yeast cells with and without Spc1. Our data show that the abundance of the membrane proteome in yeast cells lacking Spc1 is in general reduced compared to that in wild-type cells, implicating its role in controlling the cellular levels of membrane proteins.

Keywords:  Membrane proteins; Quality control; Quantity control; SPCS1; Spc12

DOI:  https://doi.org/10.1007/s00232-024-00312-5
Nat Commun. 2024 Apr 16. 15(1): 3285

Tertiary structure and conformational dynamics of the anti-amyloidogenic chaperone DNAJB6b at atomistic resolution.

Vasista Adupa, Elizaveta Ustyantseva, Harm H Kampinga, Patrick R Onck.

DNAJB6b is a molecular chaperone of the heat shock protein network, shown to play a crucial role in preventing aggregation of several disease-related intrinsically disordered proteins. Using homology modeling and microsecond-long all-atom molecular dynamics (MD) simulations, we show that monomeric DNAJB6b is a transiently interconverting protein cycling between three states: a closed state, an open state (both abundant), and a less abundant extended state. Interestingly, the reported regulatory autoinhibitory anchor between helix V in the G/F1 region and helices II/III of the J-domain, which obstructs the access of Hsp70 to the J-domain remains present in all three states. This possibly suggests a mechanistically intriguing regulation in which DNAJB6b only becomes exposed when loaded with substrates that require Hsp70 processing. Our MD results of DNAJB6b carrying mutations in the G/F1 region that are linked to limb-girdle muscular dystrophy type D1 (LGMDD1) show that this G/F1 region becomes highly dynamic, pointing towards a spontaneous release of the autoinhibitory helix V from helices II/III. This would increase the probability of non-functional Hsp70 interactions to DNAJB6b without substrates. Our cellular data indeed confirm that non-substrate loaded LGMDD1 mutants have aberrant interactions with Hsp70.

DOI: https://doi.org/10.1038/s41467-024-46587-z
Hepatology. 2024 Apr 16.

ER stress signaling at the interphase between MASH and hepatocellular carcinoma.

Younis Hazari, Eric Chevet, Béatrice Bailly-Maitre, Claudio Hetz.

Hepato-cellular carcinoma (HCC) is the most frequent primary liver cancer with an extremely poor prognosis and often develops on preset of chronic liver diseases. Major risk factors for HCC include metabolic dysfunction-associated steatohepatitis (MASH), a complex multifactorial condition associated with abnormal endoplasmic reticulum (ER) proteostasis. To cope with ER stress, the unfolded protein response (UPR) engages adaptive reactions to restore the secretory capacity of the cell. Recent advances revealed that ER stress signaling plays a critical role in HCC progression. Here we propose that chronic ER stress is a common transversal factor contributing to the transition from liver disease (risk factor) to HCC. Interventional strategies to target the UPR in HCC as cancer therapy are also discussed.

DOI: https://doi.org/10.1097/HEP.0000000000000893
J Mol Biol. 2024 Apr 16. pii: S0022-2836(24)00169-4. [Epub ahead of print] 168574

Selective autophagy of macromolecular complexes: what does it take to be taken?

Javier Lizarrondo, Florian Wilfling.

  Proteins are known to perform an astonishing array of functions thanks to their ability to cooperate and modulate each other's properties. Inside cells, proteins can assemble into large multi-subunit complexes to carry out complex cellular functions. The correct assembly and maintenance of the functional state of macromolecular protein complexes is crucial for human health. Failure to do so leads to loss of function and potential accumulation of harmful materials, which is associated with a variety of human diseases such as neurodegeneration and cancer. Autophagy engulfs cytosolic material in autophagosomes, and therefore is best suited to eliminate intact macromolecular complexes without disassembling them, which could interfere with de novo assembly. In this review, we discuss the role of autophagy in the selective degradation of macromolecular complexes. We highlight the current state of knowledge for different macromolecular complexes and their selective autophagic degradation. We emphasize the gaps in our understanding of what it takes for these large macromolecular complexes to be degraded and point to future work that may shed light on the regulation of the selective degradation of macromolecular complexes by autophagy.

Keywords:  LLPS; Selective; autophagy; cargo-recognition; phase separation; protein complexes; proteostasis

DOI:  https://doi.org/10.1016/j.jmb.2024.168574
Cell. 2024 Apr 08. pii: S0092-8674(24)00315-5. [Epub ahead of print]

Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.

Gabriela Prus, Shankha Satpathy, Brian T Weinert, Takeo Narita, Chunaram Choudhary.

  Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

Keywords:  cell signaling; dynamics; mass spectrometry; occupancy; posttranslational modification; proteomics; turnover rate; ubiquitin signaling; ubiquitylation

DOI:  https://doi.org/10.1016/j.cell.2024.03.024
iScience. 2024 Apr 19. 27(4): 109593

Type I interferon regulation by USP18 is a key vulnerability in cancer.

Veronica Jové, Heather Wheeler, Chiachin Wilson Lee, David R Healy, Kymberly Levine, Erik C Ralph, Masaya Yamaguchi, Ziyue Karen Jiang, Edward Cabral, Yingrong Xu, Jeffrey Stock, Bing Yang, Anand Giddabasappa, Paula Loria, Agustin Casimiro-Garcia, Benedikt M Kessler, Adán Pinto-Fernández, Véronique Frattini, Paul D Wes, Feng Wang.

  Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.

Keywords:  Cancer; Cell biology; Immune response; Immunity

DOI:  https://doi.org/10.1016/j.isci.2024.109593
Cell Death Dis. 2024 Apr 18. 15(4): 276

IRE1 signaling increases PERK expression during chronic ER stress.

Gideon Ong, Rosemund Ragetli, Katarzyna Mnich, Bradley W Doble, Wafa Kammouni, Susan E Logue.

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses.

DOI: https://doi.org/10.1038/s41419-024-06663-0
Cell Rep. 2024 Apr 15. pii: S2211-1247(24)00402-9. [Epub ahead of print]43(4): 114074

UPF1 regulates mRNA stability by sensing poorly translated coding sequences.

Damir Musaev, Mario Abdelmessih, Charles E Vejnar, Valeria Yartseva, Linnea A Weiss, Ethan C Strayer, Carter M Takacs, Antonio J Giraldez.

  Post-transcriptional mRNA regulation shapes gene expression, yet how cis-elements and mRNA translation interface to regulate mRNA stability is poorly understood. We find that the strength of translation initiation, upstream open reading frame (uORF) content, codon optimality, AU-rich elements, microRNA binding sites, and open reading frame (ORF) length function combinatorially to regulate mRNA stability. Machine-learning analysis identifies ORF length as the most important conserved feature regulating mRNA decay. We find that Upf1 binds poorly translated and untranslated ORFs, which are associated with a higher decay rate, including mRNAs with uORFs and those with exposed ORFs after stop codons. Our study emphasizes Upf1's converging role in surveilling mRNAs with exposed ORFs that are poorly translated, such as mRNAs with long ORFs, ORF-like 3' UTRs, and mRNAs containing uORFs. We propose that Upf1 regulation of poorly/untranslated ORFs provides a unifying mechanism of surveillance in regulating mRNA stability and homeostasis in an exon-junction complex (EJC)-independent nonsense-mediated decay (NMD) pathway that we term ORF-mediated decay (OMD).

Keywords:  CP: Molecular biology; GC-richness; NMD; ORF length; UPF1; mRNA decay; mRNA homeostasis; mRNA translation; oORF; uORF

DOI:  https://doi.org/10.1016/j.celrep.2024.114074
Curr Opin Cell Biol. 2024 Apr 15. pii: S0955-0674(24)00036-X. [Epub ahead of print]88 102357

Endoplasmic reticulum - condensate interactions in protein synthesis and secretion.

Dan T M Nguyen, Max Koppers, Ginny G Farías.

In the past decade, a growing amount of evidence has demonstrated that organelles do not act autonomously and independently but rather communicate with each other to coordinate different processes for proper cellular function. With a highly extended network throughout the cell, the endoplasmic reticulum (ER) plays a central role in interorganelle communication through membrane contact sites. Here, we highlight recent evidence indicating that the ER also forms contacts with membrane-less organelles. These interactions contribute to the dynamic assembly and disassembly of condensates and controlled protein secretion. Additionally, emerging evidence suggests their involvement in mRNA localization and localized translation. We further explore exciting future directions of this emerging theme in the organelle contact site field.

DOI: https://doi.org/10.1016/j.ceb.2024.102357
Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2315018121

Structure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex.

Duc Minh Nguyen, Deanna H Rath, Dominic Devost, Darlaine Pétrin, Robert Rizk, Alan X Ji, Naveen Narayanan, Darren Yong, Andrew Zhai, Douglas A Kuntz, Maha U Q Mian, Neil C Pomroy, Alexander F A Keszei, Samir Benlekbir, Mohammad T Mazhab-Jafari, John L Rubinstein, Terence E Hébert, Gilbert G Privé.

  Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gβ1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gβγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gβ and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gβγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

Keywords:  BTB proteins; G proteins; cryo electron microscopy; ubiquitin ligase

DOI:  https://doi.org/10.1073/pnas.2315018121
Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2317680121

A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation.

Anastasia Knyazeva, Shuang Li, Dale P Corkery, Kasturika Shankar, Laura K Herzog, Xuepei Zhang, Birendra Singh, Georg Niggemeyer, David Grill, Jonathan D Gilthorpe, Massimiliano Gaetani, Lars-Anders Carlson, Herbert Waldmann, Yao-Wen Wu.

  The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

Keywords:  ESCRT; IST1-CHMP1B; Tantalosin; endosomal recycling; noncanonical LC3 lipidation

DOI:  https://doi.org/10.1073/pnas.2317680121
Blood Adv. 2024 Apr 19. pii: bloodadvances.2023011432. [Epub ahead of print]

Lysosomal Degradation Targets Mutant Calreticulin and the Thrombopoietin Receptor in Myeloproliferative Neoplasms.

Amanpreet Kaur, Arunkumar Venkatesan, Malathi Kandarpa, Moshe Talpaz, Malini Raghavan.

Somatic mutants of calreticulin (CRT) drive myeloproliferative neoplasms (MPNs) via binding to the thrombopoietin receptor (MPL) and aberrant activation of the JAK/STAT pathway. Compared with healthy donors, platelets from MPN patients with CRT mutations display low cell surface MPL. Additionally, co-expression of MPL with an MPN-linked CRT mutant (CRTDel52) reduces cell surface MPL, suggesting that CRTDel52 may induce MPL degradation. We show that lysosomal degradation is relevant to the turnover of CRTDel52 and MPL. Furthermore, CRTDel52 increases the lysosomal localization and degradation of MPL. Mammalian target of rapamycin (mTOR) inhibitors reduce cellular CRTDel52, MPL and secreted CRTDel52 levels, and impair CRTDel52-mediated cell proliferation. mTOR inhibition also reduces colony formation and differentiation of CD34+ cells from MPN patients but not healthy donors. Together, these findings indicate low surface MPL as a biomarker of mutant CRT-mediated MPN and induced degradation of CRTDel52 and MPL as an avenue for therapeutic intervention.

DOI: https://doi.org/10.1182/bloodadvances.2023011432
Structure. 2024 Apr 12. pii: S0969-2126(24)00095-9. [Epub ahead of print]

Molecular basis for pH sensing in the KDEL trafficking receptor.

Zhiyi Wu, Kathryn Smith, Andreas Gerondopoulos, Tomoaki Sobajima, Joanne L Parker, Francis A Barr, Simon Newstead, Philip C Biggin.

  Trafficking receptors control protein localization through the recognition of specific signal sequences that specify unique cellular locations. Differences in luminal pH are important for the vectorial trafficking of cargo receptors. The KDEL receptor is responsible for maintaining the integrity of the ER by retrieving luminally localized folding chaperones in a pH-dependent mechanism. Structural studies have revealed the end states of KDEL receptor activation and the mechanism of selective cargo binding. However, precisely how the KDEL receptor responds to changes in luminal pH remains unclear. To explain the mechanism of pH sensing, we combine analysis of X-ray crystal structures of the KDEL receptor at neutral and acidic pH with advanced computational methods and cell-based assays. We show a critical role for ordered water molecules that allows us to infer a direct connection between protonation in different cellular compartments and the consequent changes in the affinity of the receptor for cargo.

Keywords:  Golgi; QM; X-ray crystallography; cargo; computational; endoplasmic reticulum; grand canonical Monte Carlo; simulation

DOI:  https://doi.org/10.1016/j.str.2024.03.013
bioRxiv. 2024 Apr 03. pii: 2024.04.03.587922. [Epub ahead of print]

Molecular characterization of Rft1, a membrane protein associated with congenital disorder of glycosylation type 1N.

Eri Hirata, Ken-Taro Sakata, Grace I Dearden, Indu Menon, George N Chiduza, Anant K Menon.

The oligosaccharide needed for protein N -glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including CDG 1N which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what role Rft1 plays in N -glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N -glycosylated. The majority of CDG 1N mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

DOI: https://doi.org/10.1101/2024.04.03.587922
Nat Protoc. 2024 Apr 18.

Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells.

Martin P Schwalm, Krishna Saxena, Susanne Müller, Stefan Knapp.

The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase. A collection of live-cell assays were designed to probe the most critical steps of the degradation process while minimizing the number of required expression constructs, making the proposed assay pipeline flexible and adaptable to the requirements of the users. This approach evaluates the underlying mechanism of selective target degraders and reveals the exact characteristics of the developed degrader molecules in living cells. The protocol allows scientists trained in basic cell culture and molecular biology to carry out small-molecule proximity-inducer screening via tracking of the ternary complex formation within 2 weeks of establishment, while degrader screening using the HiBiT system requires a CRISPR-Cas9 engineered cell line whose generation can take up to 3 months. After cell-line generation, degrader screening and validation can be carried out in high-throughput manner within days.

DOI: https://doi.org/10.1038/s41596-024-00979-z
Trends Neurosci. 2024 Apr 12. pii: S0166-2236(24)00058-4. [Epub ahead of print]

Orchestrating neuronal activity-dependent translation via the integrated stress response protein GADD34.

Xingzhi He, Wenwen Li, Huan Ma.

  In a recent study, Oliveira and colleagues revealed how growth arrest and DNA damage-inducible protein 34 (GADD34), an effector of the integrated stress response, initiates the translation of synaptic plasticity-related mRNAs following brain-derived neurotrophic factor (BDNF) stimulation. This work suggests that GADD34 may link transcriptional products with translation control upon neuronal activation, illuminating how protein synthesis is orchestrated in neuronal plasticity.

Keywords:  BDNF; excitation–transcription coupling; neuronal plasticity; protein synthesis; transcription; translation

DOI:  https://doi.org/10.1016/j.tins.2024.03.008
Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2320934121

Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.

Lijie Gu, Yanhong Du, Jianglei Chen, Mohammad Nazmul Hasan, Yung Dai Clayton, David J Matye, Jacob E Friedman, Tiangang Li.

  Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.

Keywords:  cullin; fatty liver; insulin resistance; neddylation; reductive stress

DOI:  https://doi.org/10.1073/pnas.2320934121
Nat Commun. 2024 Apr 17. 15(1): 3290

Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies.

Ying Zhu, Kerem Can Akkaya, Julia Ruta, Nanako Yokoyama, Cong Wang, Max Ruwolt, Diogo Borges Lima, Martin Lehmann, Fan Liu.

The functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome. Biochemical and imaging-based follow-up studies confirm that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. We also validate the CLASP concept in synaptic vesicles, demonstrating its applicability to different sub-cellular compartments. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, and establishes a method for concomitant profiling of sub-organelle and membrane proteomes.

DOI: https://doi.org/10.1038/s41467-024-47569-x
Nat Commun. 2024 Apr 17. 15(1): 3296

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis.

Victor E Cruz, Christine S Weirich, Nagesh Peddada, Jan P Erzberger.

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

DOI: https://doi.org/10.1038/s41467-024-47616-7
Cell Rep. 2024 Apr 12. pii: S2211-1247(24)00423-6. [Epub ahead of print]43(4): 114095

Viral infection and spread are inhibited by the polyubiquitination and downregulation of TRPV2 channel by the interferon-stimulated gene TRIM21.

Yu-Yao Guo, Yue Gao, Yun-Lin Zhao, Chang Xie, Hu Gan, Xufeng Cheng, Li-Ping Yang, Junyan Hu, Hong-Bing Shu, Bo Zhong, Dandan Lin, Jing Yao.

  Interferon (IFN) contributes to the host's antiviral response by inducing IFN-stimulated genes (ISGs). However, their functional targets and the mechanism of action remain elusive. Here, we report that one such ISG, TRIM21, interacts with and degrades the TRPV2 channel in myeloid cells, reducing its expression and providing host protection against viral infections. Moreover, viral infection upregulates TRIM21 in paracrine and autocrine manners, downregulating TRPV2 in neighboring cells to prevent viral spread to uninfected cells. Consistently, the Trim21-/- mice are more susceptible to HSV-1 and VSV infection than the Trim21+/+ littermates, in which viral susceptibility is rescued by inhibition or deletion of TRPV2. Mechanistically, TRIM21 catalyzes the K48-linked ubiquitination of TRPV2 at Lys295. TRPV2K295R is resistant to viral-infection-induced TRIM21-dependent ubiquitination and degradation, promoting viral infection more profoundly than wild-type TRPV2 when reconstituted into Lyz2-Cre;Trpv2fl/fl myeloid cells. These findings characterize targeting the TRIM21-TRPV2 axis as a conducive strategy to control viral spread to bystander cells.

Keywords:  CP: Immunology; CP: Microbiology; Ca(2+) signal; TRIM21; TRPV2; interferon-stimulated gene; plasma membrane tension; ubiquitination; viral penetration

DOI:  https://doi.org/10.1016/j.celrep.2024.114095
Cell Stem Cell. 2024 Apr 12. pii: S1934-5909(24)00096-1. [Epub ahead of print]

Nuclear RNA homeostasis promotes systems-level coordination of cell fate and senescence.

Xue Han, Linqing Xing, Yantao Hong, Xuechun Zhang, Bo Hao, J Yuyang Lu, Mengyuan Huang, Zuhui Wang, Shaoqian Ma, Ge Zhan, Tong Li, Xiaowen Hao, Yibing Tao, Guanwen Li, Shuqin Zhou, Zheng Zheng, Wen Shao, Yitian Zeng, Dacheng Ma, Wenhao Zhang, Zhen Xie, Haiteng Deng, Jiangwei Yan, Wulan Deng, Xiaohua Shen.

  Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed that RNA exosome depletion in embryonic stem cells significantly affects the transcriptome and proteome, causing pluripotency loss and pre-senescence onset. Mechanistically, exosome depletion triggers acute nuclear RNA aggregation, disrupting nuclear RNA-protein equilibrium. This disturbance limits nuclear protein availability and hinders polymerase initiation and engagement, reducing gene transcription. Concurrently, it promptly disrupts nucleolar transcription, ribosomal processes, and nuclear exporting, resulting in a translational shutdown. Prolonged exosome depletion induces nuclear structural changes resembling senescent cells, including aberrant chromatin compaction, chromocenter disassembly, and intensified heterochromatic foci. These effects suggest that the dynamic turnover of nuclear RNA orchestrates crosstalk between essential processes to optimize cellular function. Disruptions in nuclear RNA homeostasis result in systemic functional decline, altering the cell state and promoting senescence.

Keywords:  RNA exosome; RNP mesh; chromatin organization; nuclear RNA homeostasis; pluripotency; senescence; transcription; translation

DOI:  https://doi.org/10.1016/j.stem.2024.03.015
Nat Microbiol. 2024 Apr 17.

tRNA modification reprogramming contributes to artemisinin resistance in Plasmodium falciparum.

Jennifer L Small-Saunders, Ameya Sinha, Talia S Bloxham, Laura M Hagenah, Guangxin Sun, Peter R Preiser, Peter C Dedon, David A Fidock.

Plasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm5s2U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s2U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s2U reprogramming that ART-resistant parasites may employ to survive ART-induced stress.

DOI: https://doi.org/10.1038/s41564-024-01664-3
Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2317402121

Microglia-derived extracellular vesicles trigger age-related neurodegeneration upon DNA damage.

Ermioni S Arvanitaki, Evi Goulielmaki, Katerina Gkirtzimanaki, George Niotis, Edisona Tsakani, Electra Nenedaki, Iliana Rouska, Mary Kefalogianni, Dionysios Xydias, Ilias Kalafatakis, Sotiris Psilodimitrakopoulos, Domna Karagogeos, Björn Schumacher, Emmanuel Stratakis, George A Garinis.

  DNA damage and neurodegenerative disorders are intimately linked but the underlying mechanism remains elusive. Here, we show that persistent DNA lesions in tissue-resident macrophages carrying an XPF-ERCC1 DNA repair defect trigger neuroinflammation and neuronal cell death in mice. We find that microglia accumulate dsDNAs and chromatin fragments in the cytosol, which are sensed thereby stimulating a viral-like immune response in Er1Cx/- and naturally aged murine brain. Cytosolic DNAs are packaged into extracellular vesicles (EVs) that are released from microglia and discharge their dsDNA cargo into IFN-responsive neurons triggering cell death. To remove cytosolic dsDNAs and prevent inflammation, we developed targeting EVs to deliver recombinant DNase I to Er1Cx/- brain microglia in vivo. We show that EV-mediated elimination of cytosolic dsDNAs is sufficient to prevent neuroinflammation, reduce neuronal apoptosis, and delay the onset of neurodegenerative symptoms in Er1Cx/- mice. Together, our findings unveil a causal mechanism leading to neuroinflammation and provide a rationalized therapeutic strategy against age-related neurodegeneration.

Keywords:  DNA damage; extracellular vesicles; microglia; neurodegeneration

DOI:  https://doi.org/10.1073/pnas.2317402121