bims-proteo 2023-05-14 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–05–14
forty papers selected by
Eric Chevet, INSERM

Mechanisms of Protein Quality Control in the Endoplasmic Reticulum by a Coordinated Hsp40-Hsp70-Hsp90 System.
Newly imported proteins in mitochondria are particularly sensitive to aggregation.
Identifying E3 ligase substrates with quantitative degradation proteomics.
Sec16 and Sed4 interdependently function as interaction and localization partners at ER exit sites.
HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways.
Molecular basis of the TRAP complex function in ER protein biogenesis.
Protein quality control and aggregation in the endoplasmic reticulum: From basic to bedside.
TRIM56 coiled-coil domain structure provides insights into its E3 ligase functions.
SARS-CoV-2 Nonstructural Proteins 3 and 4 tune the Unfolded Protein Response.
An inventory of crosstalk between ubiquitination and other post-translational modifications in orchestrating cellular processes.
Molecular basis for recognition and deubiquitination of 40S ribosomes by Otu2.
3D structural human interactome reveals proteome-wide perturbations by disease mutations.
A ubiquitin fusion reporter to monitor muscle proteostasis in C. elegans.
FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.
The ribosome quality control factor Asc1 determines the fate of HSP70 mRNA on and off the ribosome.
E2 ubiquitin-conjugating enzymes, UBE2D1 and UBE2D2, regulate VEGFR2 dynamics and endothelial function.
Activation of XBP1 but not ATF6α rescues heart failure induced by persistent ER stress in medaka fish.
Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response.
Lysine deserts prevent adventitious ubiquitylation of ubiquitin-proteasome components.
TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.
Lysosomal-associated protein transmembrane 5 ameliorates non-alcoholic steatohepatitis by promoting the degradation of CDC42 in mice.
Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.
Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation.
ATG16L1 is equipped with two distinct WIPI2-binding sites to drive autophagy.
Signal peptide mimicry primes Sec61 for client-selective inhibition.
Secretory autophagy-promoted cargo exocytosis requires active RAB37.
Artificial Hsp104-mediated systems for re-localizing protein aggregates.
G-quadruplexes rescuing protein folding.
Optimal HSF1 activation in response to acute cold stress in BAT requires nuclear TXNIP.
Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface.
Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.
An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types.
Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in β cells.
eIF3 mRNA selectivity profiling reveals eIF3k as a cancer-relevant regulator of ribosome content.
mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.
Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation.
Dodecamer assembly of a metazoan AAA+ chaperone couples substrate extraction to refolding.
Mitochondrial translational defect extends lifespan in C. elegans by activating UPRmt.
A common mechanism of Sec61 translocon inhibition by small molecules.
Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.

Annu Rev Biophys. 2023 05 09. 52 509-524

Mechanisms of Protein Quality Control in the Endoplasmic Reticulum by a Coordinated Hsp40-Hsp70-Hsp90 System.

Judy L M Kotler, Timothy O Street.

  The Hsp40, Hsp70, and Hsp90 chaperone families are ancient, highly conserved, and critical to cellular protein homeostasis. Hsp40 chaperones can transfer their protein clients to Hsp70, and Hsp70 can transfer clients to Hsp90, but the functional benefits of these transfers are unclear. Recent structural and mechanistic work has opened up the possibility of uncovering how Hsp40, Hsp70, and Hsp90 work together as unified system. In this review, we compile mechanistic data on the ER J-domain protein 3 (ERdj3) (an Hsp40), BiP (an Hsp70), and Grp94 (an Hsp90) chaperones within the endoplasmic reticulum; what is known about how these chaperones work together; and gaps in this understanding. Using calculations, we examine how client transfer could impact the solubilization of aggregates, the folding of soluble proteins, and the triage decisions by which proteins are targeted for degradation. The proposed roles of client transfer among Hsp40-Hsp70-Hsp90 chaperones are new hypotheses, and we discuss potential experimental tests of these ideas.

Keywords:  endoplasmic reticulum; molecular chaperones; protein degradation; protein folding

DOI:  https://doi.org/10.1146/annurev-biophys-111622-091309
Acta Physiol (Oxf). 2023 May 12. e13985

Newly imported proteins in mitochondria are particularly sensitive to aggregation.

Carmela Vazquez-Calvo, Verena Kohler, Johanna L Höög, Sabrina Büttner, Martin Ott.

   AIM: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation.
METHODS: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol, using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates.
RESULTS: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation.
CONCLUSION: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.

Keywords:  Ageing; Aggregates; Cellular stress; Hsp78; Metabolism; Mitochondria; Protein quality control; Proteostasis

DOI:  https://doi.org/10.1111/apha.13985
Chembiochem. 2023 May 11. e202300108

Identifying E3 ligase substrates with quantitative degradation proteomics.

Victoria N Jordan, Alban Ordureau, Heeseon An.

  Controlled protein degradation by the ubiquitinproteasome pathway is critical for almost all cellular processes. E3 ubiquitin ligases are responsible for targeting proteins for ubiquitylation and subsequent proteasomal degradation with spatial and temporal precision. While studies have revealed various E3-substrate pairs involved in distinct biological processes, the complete substrate profiles of individual E3 ligases are largely unknown. Here we report a new approach to identify substrates of an E3 ligase for proteasomal degradation using unnatural amino acid incorporation pulse-chase proteomics (degradomics). Applying this approach, we determine the steadystate substrates of the C - t erminal to L is H (CTLH) E3 ligase, a multicomponent complex with poorly defined substrates. By comparing the proteome degradation profiles of active and inactive CTLH-expressing cells, we successfully identify previously known and new potential substrates of CTLH ligase. Altogether, degradomics can comprehensively identify degradation substrates of an E3 ligase, which can be adapted for other E3 ligases in various cellular contexts.

Keywords:  Ubiquitin, The Ubiquitin Proteasome System, Protein degradation, Protein half-life, CTLH

DOI:  https://doi.org/10.1002/cbic.202300108
J Cell Sci. 2023 May 01. pii: jcs261094. [Epub ahead of print]136(9):

Sec16 and Sed4 interdependently function as interaction and localization partners at ER exit sites.

Tomohiro Yorimitsu, Ken Sato.

  COPII proteins assemble at ER exit sites (ERES) to form transport carriers. The initiation of COPII assembly in the yeast Saccharomyces cerevisiae is triggered by the ER membrane protein Sec12. Sec16, which plays a critical role in COPII organization, localizes to ERES independently of Sec12. However, the mechanism underlying Sec16 localization is poorly understood. Here, we show that a Sec12 homolog, Sed4, is concentrated at ERES and mediates ERES localization of Sec16. We found that the interaction between Sec16 and Sed4 ensures their correct localization to ERES. Loss of the interaction with Sec16 leads to redistribution of Sed4 from the ERES specifically to high-curvature ER areas, such as the tubules and edges of the sheets. The luminal domain of Sed4 mediates this distribution, which is required for Sed4, but not for Sec16, to be concentrated at ERES. We further show that the luminal domain and its O-mannosylation are involved in the self-interaction of Sed4. Our findings provide insight into how Sec16 and Sed4 function interdependently at ERES.

Keywords:  COPII; ER; ER exit sites; Endoplasmic reticulum; Sec16; Sed4

DOI:  https://doi.org/10.1242/jcs.261094
Cell Rep. 2023 May 09. pii: S2211-1247(23)00507-7. [Epub ahead of print]42(5): 112496

HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways.

Julie K Monda, Xuezhen Ge, Moritz Hunkeler, Katherine A Donovan, Michelle W Ma, Cyrus Y Jin, Marilyn Leonard, Eric S Fischer, Eric J Bennett.

  HUWE1 is a large, enigmatic HECT-domain ubiquitin ligase implicated in the regulation of diverse pathways, including DNA repair, apoptosis, and differentiation. How HUWE1 engages its structurally diverse substrates and how HUWE1 activity is regulated are unknown. Using unbiased quantitative proteomics, we find that HUWE1 targets substrates in a largely cell-type-specific manner. However, we identify C16orf72/HAPSTR1 as a robust HUWE1 substrate in multiple cell lines. Previously established physical and genetic interactions between HUWE1 and HAPSTR1 suggest that HAPSTR1 positively regulates HUWE1 function. Here, we show that HAPSTR1 is required for HUWE1 nuclear localization and nuclear substrate targeting. Nuclear HUWE1 is required for both cell proliferation and modulation of stress signaling pathways, including p53 and nuclear factor κB (NF-κB)-mediated signaling. Combined, our results define a role for HAPSTR1 in gating critical nuclear HUWE1 functions.

Keywords:  CP: Cell biology; CP: Molecular biology; HAPSTR1/TAPR1/C16orf72; HUWE1; protein quality control; stress signaling; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2023.112496
Nat Struct Mol Biol. 2023 May 11.

Molecular basis of the TRAP complex function in ER protein biogenesis.

Mateusz Jaskolowski, Ahmad Jomaa, Martin Gamerdinger, Sandeep Shrestha, Marc Leibundgut, Elke Deuerling, Nenad Ban.

The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

DOI: https://doi.org/10.1038/s41594-023-00990-0
Front Cell Dev Biol. 2023 ;11 1156152

Protein quality control and aggregation in the endoplasmic reticulum: From basic to bedside.

Guofang Chen, Tingyi Wei, Furong Ju, Haisen Li.

  Endoplasmic reticulum (ER) is the largest membrane-bound compartment in all cells and functions as a key regulator in protein biosynthesis, lipid metabolism, and calcium balance. Mammalian endoplasmic reticulum has evolved with an orchestrated protein quality control system to handle defective proteins and ensure endoplasmic reticulum homeostasis. Nevertheless, the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum may occur during pathological conditions. The inability of endoplasmic reticulum quality control system to clear faulty proteins and aggregates from the endoplasmic reticulum results in the development of many human disorders. The efforts to comprehensively understand endoplasmic reticulum quality control network and protein aggregation will benefit the diagnostics and therapeutics of endoplasmic reticulum storage diseases. Herein, we overview recent advances in mammalian endoplasmic reticulum protein quality control system, describe protein phase transition model, and summarize the approaches to monitor protein aggregation. Moreover, we discuss the therapeutic applications of enhancing endoplasmic reticulum protein quality control pathways in endoplasmic reticulum storage diseases.

Keywords:  ER; ER storage disease; phase transition; protein aggregate; protein quality control

DOI:  https://doi.org/10.3389/fcell.2023.1156152
Comput Struct Biotechnol J. 2023 ;21 2801-2808

TRIM56 coiled-coil domain structure provides insights into its E3 ligase functions.

Xiaohua Lou, Binbin Ma, Yuan Zhuang, Xiang Xiao, Laurie J Minze, Junji Xing, Zhiqiang Zhang, Xian C Li.

  Protein ubiquitination is a post-translation modification mediated by E3 ubiquitin ligases. The RING domain E3 ligases are the largest family of E3 ubiquitin ligases, they act as a scaffold, bringing the E2-ubiquitin complex and its substrate together to facilitate direct ubiquitin transfer. However, the quaternary structures of RING E3 ligases that perform ubiquitin transfer remain poorly understood. In this study, we solved the crystal structure of TRIM56, a member of the RING E3 ligase. The structure of the coiled-coil domain indicated that the two anti-parallel dimers bound together to form a tetramer at a small crossing angle. This tetramer structure allows two RING domains to exist on each side to form an active homodimer in supporting ubiquitin transfer from E2 to its nearby substrate recruited by the C-terminal domains on the same side. These findings suggest that the coiled-coil domain-mediated tetramer is a feasible scaffold for facilitating the recruitment and transfer of ubiquitin to accomplish E3 ligase activity.

Keywords:  Coiled-coil domain; Crystal structure; E3 Ubiquitin ligase; Hydrophobic interactions; TRIM56; Tetramerization

DOI:  https://doi.org/10.1016/j.csbj.2023.04.022
bioRxiv. 2023 Apr 24. pii: 2023.04.22.537917. [Epub ahead of print]

SARS-CoV-2 Nonstructural Proteins 3 and 4 tune the Unfolded Protein Response.

Jonathan P Davies, Athira Sivadas, Katherine R Keller, Richard J H Wojcikiewicz, Lars Plate.

Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis pathways during infection through activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR). The UPR regulates protein translation, increases protein folding capacity and enhances endoplasmic reticulum (ER) biogenesis to alleviate ER stress caused by accumulation of misfolded proteins. CoVs depend on host machinery to generate large amounts of viral protein and manipulate ER-derived membranes to form double-membrane vesicles (DMVs), which serve as replication sites, making the UPR a key host pathway for CoVs to hijack. Despite the importance of CoV nonstructural proteins (nsps) in mediating replication, little is known about the role of nsps in modulating the UPR. We characterized the impact of SARS-CoV-2 nsp4, which is a key driver of DMV formation, on the UPR using quantitative proteomics. We find nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological activation of the ATF6 pathway. To determine how nsp3.1 and nsp4 might tune the UPR in concert, both proteins were co-expressed demonstrating that nsp3.1 does not suppress nsp4-mediated ATF6 activation but does suppress PERK activation. A meta-analysis of SARS-CoV-2 infection proteomics data reveals a time-dependent activation of PERK protein markers early in infection, which subsequently fades. This temporal regulation suggests a role for nsps tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic PERK activation. This work furthers our understanding of CoV-host proteostasis interactions and identifies potential areas to target for anti-viral therapies.

DOI: https://doi.org/10.1101/2023.04.22.537917
iScience. 2023 May 19. 26(5): 106276

An inventory of crosstalk between ubiquitination and other post-translational modifications in orchestrating cellular processes.

Haithem Barbour, Nadine Sen Nkwe, Benjamin Estavoyer, Clémence Messmer, Mila Gushul-Leclaire, Romain Villot, Maxime Uriarte, Karine Boulay, Sari Hlayhel, Bassel Farhat, Eric Milot, Frédérick A Mallette, Salima Daou, El Bachir Affar.

  Ubiquitination is an important post-translational modification (PTM) that regulates a large spectrum of cellular processes in eukaryotes. Abnormalities in ubiquitin signaling underlie numerous human pathologies including cancer and neurodegeneration. Much progress has been made during the last three decades in understanding how ubiquitin ligases recognize their substrates and how ubiquitination is orchestrated. Several mechanisms of regulation have evolved to prevent promiscuity including the assembly of ubiquitin ligases in multi-protein complexes with dedicated subunits and specific post-translational modifications of these enzymes and their co-factors. Here, we outline another layer of complexity involving the coordinated access of E3 ligases to substrates. We provide an extensive inventory of ubiquitination crosstalk with multiple PTMs including SUMOylation, phosphorylation, methylation, acetylation, hydroxylation, prolyl isomerization, PARylation, and O-GlcNAcylation. We discuss molecular mechanisms by which PTMs orchestrate ubiquitination, thus increasing its specificity as well as its crosstalk with other signaling pathways to ensure cell homeostasis.

Keywords:  Biochemistry; Biological sciences; Cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2023.106276
Nat Commun. 2023 May 12. 14(1): 2730

Molecular basis for recognition and deubiquitination of 40S ribosomes by Otu2.

Ken Ikeuchi, Nives Ivic, Robert Buschauer, Jingdong Cheng, Thomas Fröhlich, Yoshitaka Matsuo, Otto Berninghausen, Toshifumi Inada, Thomas Becker, Roland Beckmann.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.

DOI: https://doi.org/10.1038/s41467-023-38161-w
bioRxiv. 2023 Apr 25. pii: 2023.04.24.538110. [Epub ahead of print]

3D structural human interactome reveals proteome-wide perturbations by disease mutations.

Dapeng Xiong, Junfei Zhao, Yunguang Qiu, Yadi Zhou, Dongjin Lee, Shobhita Gupta, Weiqiang Lu, Siqi Liang, Jin Joo Kang, Charis Eng, Joseph Loscalzo, Feixiong Cheng, Haiyuan Yu.

Human genome sequencing studies have identified numerous loci associated with complex diseases. However, translating human genetic and genomic findings to disease pathobiology and therapeutic discovery remains a major challenge at multiscale interactome network levels. Here, we present a deep-learning-based ensemble framework, termed PIONEER ( P rotein-protein I nteracti O n i N t E rfac E p R ediction), that accurately predicts protein binding partner-specific interfaces for all protein interactions in humans and seven other common model organisms. We demonstrate that PIONEER outperforms existing state-of-the-art methods. We further systematically validated PIONEER predictions experimentally through generating 2,395 mutations and testing their impact on 6,754 mutation-interaction pairs, confirming PIONEER-predicted interfaces are comparable in accuracy as experimentally determined interfaces using PDB co-complex structures. We show that disease-associated mutations are enriched in PIONEER-predicted protein-protein interfaces after mapping mutations from ∼60,000 germline exomes and ∼36,000 somatic genomes. We identify 586 significant protein-protein interactions (PPIs) enriched with PIONEER-predicted interface somatic mutations (termed oncoPPIs) from pan-cancer analysis of ∼11,000 tumor whole-exomes across 33 cancer types. We show that PIONEER-predicted oncoPPIs are significantly associated with patient survival and drug responses from both cancer cell lines and patient-derived xenograft mouse models. We identify a landscape of PPI-perturbing tumor alleles upon ubiquitination by E3 ligases, and we experimentally validate the tumorigenic KEAP1-NRF2 interface mutation p.Thr80Lys in non-small cell lung cancer. We show that PIONEER-predicted PPI-perturbing alleles alter protein abundance and correlates with drug responses and patient survival in colon and uterine cancers as demonstrated by proteogenomic data from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium. PIONEER, implemented as both a web server platform and a software package, identifies functional consequences of disease-associated alleles and offers a deep learning tool for precision medicine at multiscale interactome network levels.

DOI: https://doi.org/10.1101/2023.04.24.538110
MicroPubl Biol. 2023 ;2023

A ubiquitin fusion reporter to monitor muscle proteostasis in C. elegans.

Carl Elias Kutzner, Karen Carolyn Bauer, Thorsten Hoppe.

Muscle is a highly dynamic tissue in which a variety of folding and degradation processes are active to maintain protein homeostasis (proteostasis) and functionality. The muscle-specific chaperone UNC-45 folds the motor protein myosin and assembles it into myofilaments. Malfunction of this chaperone leads to misfolding of myosin, disorganization of myofilaments, and degradation of misfolded myosin molecules by the proteasome. Here, we present a new muscle-specific ubiquitin fusion degradation (UFD) model substrate in C. elegans that helps clarify how UNC-45 dysfunction affects muscle proteostasis.

DOI: https://doi.org/10.17912/micropub.biology.000824
EMBO J. 2023 May 10. e112767

FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

Giang Thanh Nguyen-Dien, Keri-Lyn Kozul, Yi Cui, Brendan Townsend, Prajakta Gosavi Kulkarni, Soo Siang Ooi, Antonio Marzio, Nissa Carrodus, Steven Zuryn, Michele Pagano, Robert G Parton, Michael Lazarou, S Sean Millard, Robert W Taylor, Brett M Collins, Mathew Jk Jones, Julia K Pagan.

  To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

Keywords:  BNIP3; FBXL4; NIX/BNIP3L; mitochondria; mitophagy

DOI:  https://doi.org/10.15252/embj.2022112767
Nucleic Acids Res. 2023 May 09. pii: gkad338. [Epub ahead of print]

The ribosome quality control factor Asc1 determines the fate of HSP70 mRNA on and off the ribosome.

Lokha R Alagar Boopathy, Emma Beadle, Alan RuoChen Xiao, Aitana Garcia-Bueno Rico, Celia Alecki, Irene Garcia de-Andres, Kyla Edelmeier, Luca Lazzari, Mehdi Amiri, Maria Vera.

Cells survive harsh environmental conditions by potently upregulating molecular chaperones such as heat shock proteins (HSPs), particularly the inducible members of the HSP70 family. The life cycle of HSP70 mRNA in the cytoplasm is unique-it is translated during stress when most cellular mRNA translation is repressed and rapidly degraded upon recovery. Contrary to its 5' untranslated region's role in maximizing translation, we discovered that the HSP70 coding sequence (CDS) suppresses its translation via the ribosome quality control (RQC) mechanism. The CDS of the most inducible Saccharomyces cerevisiae HSP70 gene, SSA4, is uniquely enriched with low-frequency codons that promote ribosome stalling during heat stress. Stalled ribosomes are recognized by the RQC components Asc1p and Hel2p and two novel RQC components, the ribosomal proteins Rps28Ap and Rps19Bp. Surprisingly, RQC does not signal SSA4 mRNA degradation via No-Go-Decay. Instead, Asc1p destabilizes SSA4 mRNA during recovery from heat stress by a mechanism independent of ribosome binding and SSA4 codon optimality. Therefore, Asc1p operates in two pathways that converge to regulate the SSA4 mRNA life cycle during stress and recovery. Our research identifies Asc1p as a critical regulator of the stress response and RQC as the mechanism tuning HSP70 synthesis.

DOI: https://doi.org/10.1093/nar/gkad338
J Cell Sci. 2023 May 09. pii: jcs.260657. [Epub ahead of print]

E2 ubiquitin-conjugating enzymes, UBE2D1 and UBE2D2, regulate VEGFR2 dynamics and endothelial function.

William R Critchley, Gina A Smith, Ian C Zachary, Michael A Harrison, Sreenivasan Ponnambalam.

  Vascular endothelial growth factor receptor 2 (VEGFR2) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination which programs this receptor for trafficking and proteolysis but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen of the human E2 family of ubiquitin-conjugating enzymes to identify gene products which regulate VEGFR2 ubiquitination and proteolysis. We find that depletion of either UBE2D1 or UBE2D2 in endothelial cells cause a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impact on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase C1, and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell surface biotinylation and recycling studies show an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulates endothelial tubulogenesis which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis.

Keywords:  Angiogenesis; Endothelial; Signalling; UBE2D1; UBE2D2; Ubiquitin; VEGFR2

DOI:  https://doi.org/10.1242/jcs.260657
Life Sci Alliance. 2023 Jul;pii: e202201771. [Epub ahead of print]6(7):

Activation of XBP1 but not ATF6α rescues heart failure induced by persistent ER stress in medaka fish.

Byungseok Jin, Tokiro Ishikawa, Makoto Kashima, Rei Komura, Hiromi Hirata, Tetsuya Okada, Kazutoshi Mori.

The unfolded protein response is triggered in vertebrates by ubiquitously expressed IRE1α/β (although IRE1β is gut-specific in mice), PERK, and ATF6α/β, transmembrane-type sensor proteins in the ER, to cope with ER stress, the accumulation of unfolded and misfolded proteins in the ER. Here, we burdened medaka fish, a vertebrate model organism, with ER stress persistently from fertilization by knocking out the AXER gene encoding an ATP/ADP exchanger in the ER membrane, leading to decreased ATP concentration-mediated impairment of the activity of Hsp70- and Hsp90-type molecular chaperones in the ER lumen. ER stress and apoptosis were evoked from 4 and 6 dpf, respectively, leading to the death of all AXER-KO medaka by 12 dpf because of heart failure (medaka hatch at 7 dpf). Importantly, constitutive activation of IRE1α signaling-but not ATF6α signaling-rescued this heart failure and allowed AXER-KO medaka to survive 3 d longer, likely because of XBP1-mediated transcriptional induction of ER-associated degradation components. Thus, activation of a specific pathway of the unfolded protein response can cure defects in a particular organ.

DOI: https://doi.org/10.26508/lsa.202201771
Elife. 2023 May 09. pii: e79444. [Epub ahead of print]12

Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response.

Rania Garde, Abhyudai Singh, Asif Ali, David Pincus.

  The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) - rather than unfolded mature proteins - and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

Keywords:  S. cerevisiae; cell biology; computational biology; systems biology

DOI:  https://doi.org/10.7554/eLife.79444
Cell Mol Life Sci. 2023 May 09. 80(6): 143

Lysine deserts prevent adventitious ubiquitylation of ubiquitin-proteasome components.

Caroline Kampmeyer, Martin Grønbæk-Thygesen, Nicole Oelerich, Michael H Tatham, Matteo Cagiada, Kresten Lindorff-Larsen, Wouter Boomsma, Kay Hofmann, Rasmus Hartmann-Petersen.

  In terms of its relative frequency, lysine is a common amino acid in the human proteome. However, by bioinformatics we find hundreds of proteins that contain long and evolutionarily conserved stretches completely devoid of lysine residues. These so-called lysine deserts show a high prevalence in intrinsically disordered proteins with known or predicted functions within the ubiquitin-proteasome system (UPS), including many E3 ubiquitin-protein ligases and UBL domain proteasome substrate shuttles, such as BAG6, RAD23A, UBQLN1 and UBQLN2. We show that introduction of lysine residues into the deserts leads to a striking increase in ubiquitylation of some of these proteins. In case of BAG6, we show that ubiquitylation is catalyzed by the E3 RNF126, while RAD23A is ubiquitylated by E6AP. Despite the elevated ubiquitylation, mutant RAD23A appears stable, but displays a partial loss of function phenotype in fission yeast. In case of UBQLN1 and BAG6, introducing lysine leads to a reduced abundance due to proteasomal degradation of the proteins. For UBQLN1 we show that arginine residues within the lysine depleted region are critical for its ability to form cytosolic speckles/inclusions. We propose that selective pressure to avoid lysine residues may be a common evolutionary mechanism to prevent unwarranted ubiquitylation and/or perhaps other lysine post-translational modifications. This may be particularly relevant for UPS components as they closely and frequently encounter the ubiquitylation machinery and are thus more susceptible to nonspecific ubiquitylation.

Keywords:  Degradation; Intrinsically disordered protein; Lysine; PTM; Proteasome; Ubiquitin

DOI:  https://doi.org/10.1007/s00018-023-04782-z
Cell Rep. 2023 Apr 30. pii: S2211-1247(23)00465-5. [Epub ahead of print] 112454

TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.

Shiori Akabane, Kiyona Watanabe, Hidetaka Kosako, Shun-Ichi Yamashita, Kohei Nishino, Masahiro Kato, Shiori Sekine, Tomotake Kanki, Noriyuki Matsuda, Toshiya Endo, Toshihiko Oka.

  PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.

Keywords:  CP: Cell biology; OMA1; PINK1; TIM23; mitochondrial quality control

DOI:  https://doi.org/10.1016/j.celrep.2023.112454
Nat Commun. 2023 May 08. 14(1): 2654

Lysosomal-associated protein transmembrane 5 ameliorates non-alcoholic steatohepatitis by promoting the degradation of CDC42 in mice.

Lang Jiang, Jing Zhao, Qin Yang, Mei Li, Hao Liu, Xiaoyue Xiao, Song Tian, Sha Hu, Zhen Liu, Peiwen Yang, Manhua Chen, Ping Ye, Jiahong Xia.

Non-alcoholic steatohepatitis (NASH) has received great attention due to its high incidence. Here, we show that lysosomal-associated protein transmembrane 5 (LAPTM5) is associated with NASH progression through extensive bioinformatical analysis. The protein level of LAPTM5 bears a negative correlation with NAS score. Moreover, LAPTM5 degradation is mediated through its ubiquitination modification by the E3 ubquitin ligase NEDD4L. Discovered by experiments conducted on male mice, hepatocyte-specific depletion of Laptm5 exacerbates mouse NASH symptoms. In contrast, Laptm5 overexpression in hepatocytes exerts diametrically opposite effects. Mechanistically, LAPTM5 interacts with CDC42 and promotes its degradation through a lysosome-dependent manner under the stimulation of palmitic acid, thus inhibiting activation of the mitogen-activated protein kinase signaling pathway. Finally, adenovirus-mediated hepatic Laptm5 overexpression ameliorates aforementioned symptoms in NASH models.

DOI: https://doi.org/10.1038/s41467-023-37908-9
Protein Sci. 2023 May 11. e4656

Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.

Vanessa Nguyen, Ethan Ahler, Katherine A Sitko, Jason J Stephany, Dustin J Maly, Douglas M Fowler.

Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3,929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and β1-β2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and β1-β2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1002/pro.4656
Commun Biol. 2023 May 08. 6(1): 497

Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation.

Gefei Chen, Axel Leppert, Helen Poska, Harriet E Nilsson, Carlos Piedrafita Alvira, Xueying Zhong, Philip Koeck, Caroline Jegerschöld, Axel Abelein, Hans Hebert, Jan Johansson.

ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.

DOI: https://doi.org/10.1038/s42003-023-04883-2
Autophagy. 2023 May 10.

ATG16L1 is equipped with two distinct WIPI2-binding sites to drive autophagy.

Xinyu Gong, Lifeng Pan.

  The recruitment of ATG12-ATG5-ATG16L1 complex to phagophore mediated by the specific interaction between ATG16L1 and WIPI2, is pivotal to the formation of autophagosomes during macroautophagy. Recently, we reported that ATG16L1 contains two distinct WIPI2-binding sites, the previously reported WIPI2-binding site (WBS1), and the newly identified site (WBS2). By determining the crystal structures of WIPI2 with ATG16L1 WBS1 and WBS2 respectively, we uncovered that, unlike ATG16L1 WBS1, ATG16L1 WBS2 and its binding mechanism to WIPI2 are conserved from yeast to mammals. Using cell-based functional assays, we further demonstrated that the integrity of two WIPI2-binding sites of ATG16L1 is essential for normal autophagic flux. In summary, our study provided mechanistic insights into the interaction of two key autophagic proteins, ATG16L1 and WIPI2, and revealed a dual-binding-site mode adopted by ATG16L1 to associate with WIPI2.

Keywords:  ATG16L1; Macroautophagy; WIPI2; autophagosome

DOI:  https://doi.org/10.1080/15548627.2023.2213038
Nat Chem Biol. 2023 May 11.

Signal peptide mimicry primes Sec61 for client-selective inhibition.

Shahid Rehan, Dale Tranter, Phillip P Sharp, Gregory B Craven, Eric Lowe, Janet L Anderl, Tony Muchamuel, Vahid Abrishami, Suvi Kuivanen, Nicole A Wenzell, Andy Jennings, Chakrapani Kalyanaraman, Tomas Strandin, Matti Javanainen, Olli Vapalahti, Matthew P Jacobson, Dustin McMinn, Christopher J Kirk, Juha T Huiskonen, Jack Taunton, Ville O Paavilainen.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.

DOI: https://doi.org/10.1038/s41589-023-01326-1
Autophagy. 2023 May 12. 1-2

Secretory autophagy-promoted cargo exocytosis requires active RAB37.

Shan-Ying Wu, Yi-Ching Wang, Roberto Zuchini, Kai-Ying Lan, Hsiao-Sheng Liu, Sheng-Hui Lan.

  RAB37 GTPase regulates cargo exocytosis by cycling between an inactive GDP-bound form and an active GTP-bound form. We reveal that RAB37 simultaneously regulates autophagy activation and tissue inhibitor of metalloproteinase 1 (TIMP1) secretion in lung cancer cells under starvation conditions. TIMP1, an inflammatory cytokine, is a known inhibitory molecule of matrix metalloproteinases matrix metalloproteinase 9 and suppresses the mobility of lung cancer cells both in vitro and in vivo through conventional exocytosis under serum-free conditions. Notably, we disclosed that secretory autophagy participates in TIMP1 secretion in a RAB37- and Sec22b-dependent manner. Sec22b, a SNARE family protein, participates in vesicle and membrane fusion of secretory autophagy. Knockdown of Sec22b decreased TIMP1 secretion and cell motility but did not affect cell proliferation under starvation conditions. We confirmed that starvation-activated RAB37 accompanied by Sec22b is essential for secretory autophagy to further enhance TIMP1 exocytosis. We further use an off-label drug amiodarone to demonstrate that autophagy induction facilitates TIMP1 secretion and suppresses the motility and metastasis of lung cancer cells in a RAB37-dependent manner in the lung-to-lung mouse model. In conclusion, we demonstrated that the RAB37 activation plays a pivotal regulatory role in secretory autophagy for TIMP1 secretion in lung cancer.Abbreviations: ATG: autophagy-related gene; GDP: guanosine diphosphate; GTP: guanosine triphosphate; LC3: microtubule-associated protein 1A/1B-light chain 3; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TIMP1: tissue inhibitor matrix metalloproteinase 1.

Keywords:  RAB37; TIMP1; lung cancer; metastasis; secretory autophagy

DOI:  https://doi.org/10.1080/15548627.2023.2210446
Nat Commun. 2023 May 09. 14(1): 2663

Artificial Hsp104-mediated systems for re-localizing protein aggregates.

Arthur Fischbach, Angela Johns, Kara L Schneider, Xinxin Hao, Peter Tessarz, Thomas Nyström.

Spatial Protein Quality Control (sPQC) sequesters misfolded proteins into specific, organelle-associated inclusions within the cell to control their toxicity. To approach the role of sPQC in cellular fitness, neurodegenerative diseases and aging, we report on the construction of Hsp100-based systems in budding yeast cells, which can artificially target protein aggregates to non-canonical locations. We demonstrate that aggregates of mutant huntingtin (mHtt), the disease-causing agent of Huntington's disease can be artificially targeted to daughter cells as well as to eisosomes and endosomes with this approach. We find that the artificial removal of mHtt inclusions from mother cells protects them from cell death suggesting that even large mHtt inclusions may be cytotoxic, a trait that has been widely debated. In contrast, removing inclusions of endogenous age-associated misfolded proteins does not significantly affect the lifespan of mother cells. We demonstrate also that this approach is able to manipulate mHtt inclusion formation in human cells and has the potential to be useful as an alternative, complementary approach to study the role of sPQC, for example in aging and neurodegenerative disease.

DOI: https://doi.org/10.1038/s41467-023-37706-3
Proc Natl Acad Sci U S A. 2023 05 16. 120(20): e2216308120

G-quadruplexes rescuing protein folding.

Ahyun Son, Veronica Huizar Cabral, Zijue Huang, Theodore J Litberg, Scott Horowitz.

  Maintaining the health of the proteome is a critical cellular task. Recently, we found G-quadruplex (G4) nucleic acids are especially potent at preventing protein aggregation in vitro and could at least indirectly improve the protein folding environment of Escherichia coli. However, the roles of G4s in protein folding were not yet explored. Here, through in vitro protein folding experiments, we discover that G4s can accelerate protein folding by rescuing kinetically trapped intermediates to both native and near-native folded states. Time-course folding experiments in E. coli further demonstrate that these G4s primarily improve protein folding quality in E. coli as opposed to preventing protein aggregation. The ability of a short nucleic acid to rescue protein folding opens up the possibility of nucleic acids and ATP-independent chaperones to play considerable roles in dictating the ultimate folding fate of proteins.

Keywords:  G4; protein folding; proteostasis; quadruplex

DOI:  https://doi.org/10.1073/pnas.2216308120
iScience. 2023 May 19. 26(5): 106538

Optimal HSF1 activation in response to acute cold stress in BAT requires nuclear TXNIP.

Althea N Waldhart, Kin H Lau, Holly Dykstra, Tracey Avequin, Ning Wu.

  While TXNIP (thioredoxin interacting protein) in the plasma membrane and vesicular location is known to negatively regulate cellular glucose uptake by facilitating glucose transporter endocytosis, the function of TXNIP in the nucleus is far less understood. Herein, we sought to determine the function of nuclear TXNIP in vivo, using a new HA-tagged TXNIP knock-in mouse model. We observed that TXNIP can be found in the nucleus of a variety of cells from different tissues including hepatocytes (liver), enterocytes (small intestine), exocrine cells (pancreas), and brown adipocytes (BAT). Further investigations into the role of nuclear TXNIP in BAT revealed that cold stress rapidly and transiently activated HSF1 (heat shock factor 1). HSF1 interaction with TXNIP during its activation is required for optimal HSF1 directed cold shock response in BAT.

Keywords:  Biological sciences; Molecular biology; Molecular interaction; Physiology

DOI:  https://doi.org/10.1016/j.isci.2023.106538
Nat Commun. 2023 May 09. 14(1): 2683

Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface.

Yuta Amagai, Momo Yamada, Toshiyuki Kowada, Tomomi Watanabe, Yuyin Du, Rong Liu, Satoshi Naramoto, Satoshi Watanabe, Junko Kyozuka, Tiziana Anelli, Tiziana Tempio, Roberto Sitia, Shin Mizukami, Kenji Inaba.

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

DOI: https://doi.org/10.1038/s41467-023-38397-6
bioRxiv. 2023 Apr 25. pii: 2023.04.25.537803. [Epub ahead of print]

Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

Ambre J Sala, Rogan A Grant, Ghania Imran, Claire Morton, Renee M Brielmann, Laura C Bott, Jennifer Watts, Richard I Morimoto.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in C. elegans . We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, a functional homolog of mammalian peroxisome proliferator-activated receptor alpha (PPARα), regulates stress resilience and proteostasis downstream of embryo integrity and other pathways that influence lipid homeostasis, and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing inter-tissue pathway in somatic tissues, also triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49 together with its co-activator MDT-15 contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer as well as by other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting. Further, we show that increased NHR-49 activity is sufficient to suppress polyglutamine aggregation and improve stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

DOI: https://doi.org/10.1101/2023.04.25.537803
bioRxiv. 2023 Apr 29. pii: 2023.04.28.538590. [Epub ahead of print]

An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types.

Jung-Kuei Chen, Karl A Merrick, Yi Wen Kong, Anita Izrael-Tomasevic, George Eng, Erika D Handly, Jesse C Patterson, Ian G Cannell, Lucia Suarez-Lopez, Aaron M Hosios, Anh Dinh, Donald S Kirkpatrick, Kebing Yu, Christopher M Rose, Jonathan M Hernandez, Haeun Hwangbo, Adam C Palmer, Matthew G Vander Heiden, Ömer H Yilmaz, Michael B Yaffe.

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

DOI: https://doi.org/10.1101/2023.04.28.538590
Cell Rep. 2023 May 11. pii: S2211-1247(23)00511-9. [Epub ahead of print]42(5): 112500

Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in β cells.

Julie Lacombe, Kevin Guo, Jessica Bonneau, Denis Faubert, Florian Gioanni, Alexis Vivoli, Sarah M Muir, Soraya Hezzaz, Vincent Poitout, Mathieu Ferron.

  Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse β cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced β cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in β cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in β cells and in their capacity to adapt to metabolic stress.

Keywords:  CP: Metabolism; ERGP; GGCX; diabetes; insulin secretion; store-operated calcium entry; vitamin K; β cells; γ-carboxylation

DOI:  https://doi.org/10.1016/j.celrep.2023.112500
EMBO J. 2023 May 08. e112362

eIF3 mRNA selectivity profiling reveals eIF3k as a cancer-relevant regulator of ribosome content.

Haoran Duan, Siqiong Zhang, Yoram Zarai, Rupert Öllinger, Yanmeng Wu, Li Sun, Cheng Hu, Yaohui He, Guiyou Tian, Roland Rad, Xiangquan Kong, Yabin Cheng, Tamir Tuller, Dieter A Wolf.

  eIF3, whose subunits are frequently overexpressed in cancer, regulates mRNA translation from initiation to termination, but mRNA-selective functions of individual subunits remain poorly defined. Using multiomic profiling upon acute depletion of eIF3 subunits, we observed that while eIF3a, b, e, and f markedly differed in their impact on eIF3 holo-complex formation and translation, they were each required for cancer cell proliferation and tumor growth. Remarkably, eIF3k showed the opposite pattern with depletion promoting global translation, cell proliferation, tumor growth, and stress resistance through repressing the synthesis of ribosomal proteins, especially RPS15A. Whereas ectopic expression of RPS15A mimicked the anabolic effects of eIF3k depletion, disruption of eIF3 binding to the 5'-UTR of RSP15A mRNA negated them. eIF3k and eIF3l are selectively downregulated in response to endoplasmic reticulum and oxidative stress. Supported by mathematical modeling, our data uncover eIF3k-l as a mRNA-specific module which, through controlling RPS15A translation, serves as a rheostat of ribosome content, possibly to secure spare translational capacity that can be mobilized during stress.

Keywords:  RPS15A; cancer growth control; mRNA selectivity; ribosome content; translation initiation factor 3

DOI:  https://doi.org/10.15252/embj.2022112362
bioRxiv. 2023 Apr 26. pii: 2023.04.24.538105. [Epub ahead of print]

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Alexander N Gasparski, Konstadinos Moissoglu, Sandeep Pallikkuth, Sezen Meydan, Nicholas R Guydosh, Stavroula Mili.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a 'partner-selection' mechanism that robustly influences protein distribution and function.

DOI: https://doi.org/10.1101/2023.04.24.538105
Nat Commun. 2023 May 08. 14(1): 2642

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation.

Jonathan J Swietlik, Stefanie Bärthel, Chiara Falcomatà, Diana Fink, Ankit Sinha, Jingyuan Cheng, Stefan Ebner, Peter Landgraf, Daniela C Dieterich, Henrik Daub, Dieter Saur, Felix Meissner.

Cell-selective proteomics is a powerful emerging concept to study heterocellular processes in tissues. However, its high potential to identify non-cell-autonomous disease mechanisms and biomarkers has been hindered by low proteome coverage. Here, we address this limitation and devise a comprehensive azidonorleucine labeling, click chemistry enrichment, and mass spectrometry-based proteomics and secretomics strategy to dissect aberrant signals in pancreatic ductal adenocarcinoma (PDAC). Our in-depth co-culture and in vivo analyses cover more than 10,000 cancer cell-derived proteins and reveal systematic differences between molecular PDAC subtypes. Secreted proteins, such as chemokines and EMT-promoting matrisome proteins, associated with distinct macrophage polarization and tumor stromal composition, differentiate classical and mesenchymal PDAC. Intriguingly, more than 1,600 cancer cell-derived proteins including cytokines and pre-metastatic niche formation-associated factors in mouse serum reflect tumor activity in circulation. Our findings highlight how cell-selective proteomics can accelerate the discovery of diagnostic markers and therapeutic targets in cancer.

DOI: https://doi.org/10.1038/s41467-023-38171-8
Sci Adv. 2023 May 10. 9(19): eadf5336

Dodecamer assembly of a metazoan AAA+ chaperone couples substrate extraction to refolding.

Arpit Gupta, Alfred M Lentzsch, Alex Siegel, Zanlin Yu, Un Seng Chio, Yifan Cheng, Shu-Ou Shan.

Ring-forming AAA+ chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA+ chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.

DOI: https://doi.org/10.1126/sciadv.adf5336
Redox Biol. 2023 May 07. pii: S2213-2317(23)00123-4. [Epub ahead of print]63 102722

Mitochondrial translational defect extends lifespan in C. elegans by activating UPRmt.

Miaomiao Guo, Xinhua Qiao, Yuanyuan Wang, Zi-Han Li, Chang Shi, Yun Chen, Lu Kang, Chang Chen, Xiao-Long Zhou.

  Aminoacyl-tRNA synthetases (aaRSs) are indispensable players in translation. Usually, two or three genes encode cytoplasmic and mitochondrial threonyl-tRNA synthetases (ThrRSs) in eukaryotes. Here, we reported that Caenorhabditis elegans harbors only one tars-1, generating cytoplasmic and mitochondrial ThrRSs via translational reinitiation. Mitochondrial tars-1 knockdown decreased mitochondrial tRNAThr charging and translation and caused pleotropic phenotypes of delayed development, decreased motor ability and prolonged lifespan, which could be rescued by replenishing mitochondrial tars-1. Mitochondrial tars-1 deficiency leads to compromised mitochondrial functions including the decrease in oxygen consumption rate, complex Ⅰ activity and the activation of the mitochondrial unfolded protein response (UPRmt), which contributes to longevity. Furthermore, deficiency of other eight mitochondrial aaRSs in C. elegans and five in mammal also caused activation of the UPRmt. In summary, we deciphered the mechanism of one tars-1, generating two aaRSs, and elucidated the biochemical features and physiological function of C. elegans tars-1. We further uncovered a conserved connection between mitochondrial translation deficiency and UPRmt.

Keywords:  Aminoacyl-tRNA synthetases; Lifespan; Mitochondrial translation; UPR(mt); tars-1(ora1) Ⅱ/wt

DOI:  https://doi.org/10.1016/j.redox.2023.102722
Nat Chem Biol. 2023 May 11.

A common mechanism of Sec61 translocon inhibition by small molecules.

Samuel Itskanov, Laurie Wang, Tina Junne, Rumi Sherriff, Li Xiao, Nicolas Blanchard, Wei Q Shi, Craig Forsyth, Dominic Hoepfner, Martin Spiess, Eunyong Park.

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design.

DOI: https://doi.org/10.1038/s41589-023-01337-y
Stem Cell Reports. 2023 May 09. pii: S2213-6711(23)00136-4. [Epub ahead of print]18(5): 1090-1106

Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.

Malgorzata Zatyka, Tatiana R Rosenstock, Congxin Sun, Adina M Palhegyi, Georgina W Hughes, Samuel Lara-Reyna, Dewi Astuti, Alessandro di Maio, Axel Sciauvaud, Miriam E Korsgen, Vesna Stanulovic, Gamze Kocak, Malgorzata Rak, Sandra Pourtoy-Brasselet, Katherine Winter, Thiago Varga, Margot Jarrige, Hélène Polvèche, Joao Correia, Eva-Maria Frickel, Maarten Hoogenkamp, Douglas G Ward, Laetitia Aubry, Timothy Barrett, Sovan Sarkar.

  Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.

Keywords:  Cyclosporin A; Human induced pluripotent stem cell-derived neurons; Mitochondria-associated ER membrane; Mitochondrial dysfunction; Mitochondrial membrane potential; MnTBAP; Neurodegeneration; VDAC1; WFS1; Wolfram syndrome

DOI:  https://doi.org/10.1016/j.stemcr.2023.04.002