bims-proteo 2024-04-28 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–04–28
37 papers selected by
Eric Chevet, INSERM

Baseline Unfolded Protein Response Signaling Adjusts the Timing of the Mammalian Cell Cycle.
FicD regulates adaptation to the unfolded protein response in the murine liver.
Advances in nuclear proteostasis of metazoans.
Structural Basis of Conformational Dynamics in the PROTAC-Induced Protein Degradation.
Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control.
Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy.
The Zn2+ transporter ZIP7 enhances endoplasmic-reticulum-associated protein degradation and prevents neurodegeneration in Drosophila.
Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP.
Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts.
Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.
Large-scale chemoproteomics expedites ligand discovery and predicts ligand behavior in cells.
Phage defence system CBASS is regulated by a prokaryotic E2 enzyme that imitates the ubiquitin pathway.
Erasing marks: Functions of plant deubiquitylating enzymes in modulating the ubiquitin code.
Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1β production in human macrophages and microglia.
The Wolfram-like variant WFS1E864K destabilizes MAM and compromises autophagy and mitophagy in human and mice.
Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation.
Nuclear speckle rejuvenation alleviates proteinopathies at the expense of YAP1.
Maximum entropy determination of mammalian proteome dynamics.
Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR).
Proteostatic Remodeling of Small Heat Shock Chaperones─Crystallins by Ran-Binding Protein 2─and the Peptidyl-Prolyl cis-trans Isomerase and Chaperone Activities of Its Cyclophilin Domain.
Mechanism of Ψ-Pro/C-degron recognition by the CRL2FEM1B ubiquitin ligase.
Integrative spatial analysis reveals a multi-layered organization of glioblastoma.
Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer.
The MYCN 5' UTR as a therapeutic target in neuroblastoma.
Chemical codes promote selective compartmentalization of proteins.
ANKZF1 knockdown inhibits glioblastoma progression by promoting intramitochondrial protein aggregation through mitoRQC.
STING promotes homeostatic maintenance of tissues and confers longevity with aging.
UFL1 triggers replication fork degradation by MRE11 in BRCA1/2-deficient cells.
TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.
Predictomes: A classifier-curated database of AlphaFold-modeled protein-protein interactions.
The IRE1α-XBP1 arm of the unfolded protein response is a host factor activated in SARS-CoV-2 infection.
The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.
Molecular recording of calcium signals via calcium-dependent proximity labeling.
PARP1 UFMylation ensures the stability of stalled replication forks.
Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.
AviTrap: A novel solution to achieve complete biotinylation.
The phosphorylation of carboxyl-terminal eIF2α by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis.

Mol Biol Cell. 2024 Apr 24. mbcE23110419

Baseline Unfolded Protein Response Signaling Adjusts the Timing of the Mammalian Cell Cycle.

Soham P Chowdhury, Sabrina C Solley, Elena Polishchuk, Julien Bacal, Julia E Conrad, Brooke M Gardner, Diego Acosta-Alvear, Francesca Zappa.

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2⍺ had no such effect, suggesting that cell cycle timing depends on PERK's kinase activity but is independent of eIF2⍺ phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.

DOI: https://doi.org/10.1091/mbc.E23-11-0419
bioRxiv. 2024 Apr 17. pii: 2024.04.15.589620. [Epub ahead of print]

FicD regulates adaptation to the unfolded protein response in the murine liver.

Amanda K Casey, Nathan M Stewart, Naqi Zaidi, Hillery F Gray, Amelia Cox, Hazel A Fields, Kim Orth.

The unfolded protein response (UPR) is a cellular stress response that is activated when misfolded proteins accumulate in the endoplasmic reticulum (ER). The UPR elicits a signaling cascade that results in an upregulation of protein folding machinery and cell survival signals. However, prolonged UPR responses can result in elevated cellular inflammation, damage, and even cell death. Thus, regulation of the UPR response must be tuned to the needs of the cell, sensitive enough to respond to the stress but pliable enough to be stopped after the crisis has passed. Previously, we discovered that the bi-functional enzyme FicD can modulate the UPR response via post-translational modification of BiP. FicD AMPylates BiP during homeostasis and deAMPylates BiP during stress. We found this activity is important for the physiological regulation of the exocrine pancreas. Here, we explore the role of FicD in the murine liver. Like our previous studies, livers lacking FicD exhibit enhanced UPR signaling in response to short term physiologic fasting and feeding stress. However, the livers of FicD -/- did not show marked changes in UPR signaling or damage after either chronic high fat diet (HFD) feeding or acute pathological UPR induction. Intriguingly, FicD -/- mice showed changes in UPR induction and weight loss patterns following repeated pathological UPR induction. These findings show that FicD regulates UPR responses during mild physiological stress and may play a role in maintaining resiliency of tissue through adaptation to repeated ER stress.

DOI: https://doi.org/10.1101/2024.04.15.589620
Biochimie. 2024 Apr 18. pii: S0300-9084(24)00081-6. [Epub ahead of print]

Advances in nuclear proteostasis of metazoans.

Julia Buggiani, Thierry Meinnel, Carmela Giglione, Frédéric Frottin.

  The proteostasis network and associated protein quality control (PQC) mechanisms ensure proteome functionality and are essential for cell survival. A distinctive feature of eukaryotic cells is their high degree of compartmentalization, requiring specific and adapted proteostasis networks for each compartment. The nucleus, essential for maintaining the integrity of genetic information and gene transcription, is one such compartment. While PQC mechanisms have been investigated for decades in the cytoplasm and the endoplasmic reticulum, our knowledge of nuclear PQC pathways is only emerging. Recent developments in the field have underscored the importance of spatially managing aberrant proteins within the nucleus. Upon proteotoxic stress, misfolded proteins and PQC effectors accumulate in various nuclear membrane-less organelles. Beyond bringing together effectors and substrates, the biophysical properties of these organelles allow novel PQC functions. In this review, we explore the specificity of the nuclear compartment, the effectors of the nuclear proteostasis network, and the PQC roles of nuclear membrane-less organelles in metazoans.

Keywords:  Nuclear proteostasis; chaperone; liquid-liquid phase separation; membrane-less organelle; protein degradation; protein quality control

DOI:  https://doi.org/10.1016/j.biochi.2024.04.006
ChemMedChem. 2024 Apr 24. e202400171

Structural Basis of Conformational Dynamics in the PROTAC-Induced Protein Degradation.

Hongtao Zhao.

  Pronounced conformational dynamics is unveiled upon analyzing multiple crystal structures of the same proteins recruited to the same E3 ligases by PROTACs, and yet, is largely permissive for targeted protein degradation due to the intrinsic mobility of E3 assemblies creating a large ubiquitylation zone. Mathematical modelling of ternary dynamics on ubiquitylation probability confirms the experimental finding that ternary complex rigidification need not correlate with enhanced protein degradation. Salt bridges are found to prevail in the PROTAC-induced ternary complexes, and may contribute to a positive cooperativity and prolonged half-life. The analysis highlights the importance of presenting lysines close to the active site of the E2 enzyme while constraining ternary dynamics in PROTAC design to achieve high degradation efficiency.

Keywords:  PROTAC, conformational dynamics, ubiquitylation, degradation

DOI:  https://doi.org/10.1002/cmdc.202400171
Nucleic Acids Res. 2024 Apr 22. pii: gkae285. [Epub ahead of print]

Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control.

Katharine Y Chen, Heungwon Park, Arvind Rasi Subramaniam.

Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level.

DOI: https://doi.org/10.1093/nar/gkae285
J Cell Biol. 2024 Jun 03. pii: e202306150. [Epub ahead of print]223(6):

Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy.

Madison A Ward, Janakiram R Vangala, Hatem Elif Kamber Kaya, Holly A Byers, Nayyerehalsadat Hosseini, Antonio Diaz, Ana Maria Cuervo, Susmita Kaushik, Senthil K Radhakrishnan.

Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.

DOI: https://doi.org/10.1083/jcb.202306150
Dev Cell. 2024 Apr 22. pii: S1534-5807(24)00228-4. [Epub ahead of print]

The Zn2+ transporter ZIP7 enhances endoplasmic-reticulum-associated protein degradation and prevents neurodegeneration in Drosophila.

Xiaoran Guo, Morgan Mutch, Alba Yurani Torres, Maddalena Nano, Nishi Rauth, Jacob Harwood, Drew McDonald, Zijing Chen, Craig Montell, Wei Dai, Denise J Montell.

  Proteotoxic stress drives numerous degenerative diseases. Cells initially adapt to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated protein degradation (ERAD). However, persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. The ER-localized Zn2+ transporter ZIP7 is conserved from plants to humans and required for intestinal self-renewal, Notch signaling, cell motility, and survival. However, a unifying mechanism underlying these diverse phenotypes was unknown. In studying Drosophila border cell migration, we discovered that ZIP7-mediated Zn2+ transport enhances the obligatory deubiquitination of proteins by the Rpn11 Zn2+ metalloproteinase in the proteasome lid. In human cells, ZIP7 and Zn2+ are limiting for deubiquitination. In a Drosophila model of neurodegeneration caused by misfolded rhodopsin (Rh1), ZIP7 overexpression degrades misfolded Rh1 and rescues photoreceptor viability and fly vision. Thus, ZIP7-mediated Zn2+ transport is a previously unknown, rate-limiting step for ERAD in vivo with therapeutic potential in protein misfolding diseases.

Keywords:  Drosophila; ER stress; ERAD; apoptosis; border cell migration; integrated stress response; neurodegeneration; proteasome; retinitis pigmentosa; zinc transport

DOI:  https://doi.org/10.1016/j.devcel.2024.04.003
RNA. 2024 Apr 24. pii: rna.079890.123. [Epub ahead of print]

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP.

Soon Yi, Shashi S Singh, Kathryn Rozen-Gagnon, Joseph M Luna.

  RNA binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Keywords:  CrossLinking and ImmunoPrecipitation (CLIP); HuR/ELAVL1; Proximity Labeling; RNA Binding Proteins; RNA Localization

DOI:  https://doi.org/10.1261/rna.079890.123
bioRxiv. 2024 Apr 20. pii: 2024.04.15.589678. [Epub ahead of print]

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts.

Hendrik Glauninger, Jared A M Bard, Caitlin J Wong Hickernell, Edo M Airoldi, Weihan Li, Robert H Singer, Sneha Paul, Jingyi Fei, Tobin R Sosnick, Edward W J Wallace, D Allan Drummond.

Stress-induced condensation of mRNA and proteins into stress granules is conserved across eukaryotes, yet the function, formation mechanisms, and relation to well-studied conserved transcriptional responses remain largely unresolved. Stress-induced exposure of ribosome-free mRNA following translational shutoff is thought to cause condensation by allowing new multivalent RNA-dependent interactions, with RNA length and associated interaction capacity driving increased condensation. Here we show that, in striking contrast, virtually all mRNA species condense in response to multiple unrelated stresses in budding yeast, length plays a minor role, and instead, stress-induced transcripts are preferentially excluded from condensates, enabling their selective translation. Using both endogenous genes and reporter constructs, we show that translation initiation blockade, rather than resulting ribosome-free RNA, causes condensation. These translation initiation-inhibited condensates (TIICs) are biochemically detectable even when stress granules, defined as microscopically visible foci, are absent or blocked. TIICs occur in unstressed yeast cells, and, during stress, grow before the appearance of visible stress granules. Stress-induced transcripts are excluded from TIICs primarily due to the timing of their expression, rather than their sequence features. Together, our results reveal a simple system by which cells redirect translational activity to newly synthesized transcripts during stress, with broad implications for cellular regulation in changing conditions.

DOI: https://doi.org/10.1101/2024.04.15.589678
bioRxiv. 2023 May 13. pii: 2023.05.13.540638. [Epub ahead of print]

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

Ian Cooney, Heidi L Schubert, Karina Cedeno, Hsien-Jung L Lin, John C Price, Christopher P Hill, Peter S Shen.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase cassettes, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of structures of Cdc48 affinity purified from lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data reveal new elements of Shp1-Cdc48 binding and support a "hand-over-hand" mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by D2 in substrate translocation/unfolding.

DOI: https://doi.org/10.1101/2023.05.13.540638
Science. 2024 Apr 26. 384(6694): eadk5864

Large-scale chemoproteomics expedites ligand discovery and predicts ligand behavior in cells.

Fabian Offensperger, Gary Tin, Miquel Duran-Frigola, Elisa Hahn, Sarah Dobner, Christopher W Am Ende, Joseph W Strohbach, Andrea Rukavina, Vincenth Brennsteiner, Kevin Ogilvie, Nara Marella, Katharina Kladnik, Rodolfo Ciuffa, Jaimeen D Majmudar, S Denise Field, Ariel Bensimon, Luca Ferrari, Evandro Ferrada, Amanda Ng, Zhechun Zhang, Gianluca Degliesposti, Andras Boeszoermenyi, Sascha Martens, Robert Stanton, André C Müller, J Thomas Hannich, David Hepworth, Giulio Superti-Furga, Stefan Kubicek, Monica Schenone, Georg E Winter.

Chemical modulation of proteins enables a mechanistic understanding of biology and represents the foundation of most therapeutics. However, despite decades of research, 80% of the human proteome lacks functional ligands. Chemical proteomics has advanced fragment-based ligand discovery toward cellular systems, but throughput limitations have stymied the scalable identification of fragment-protein interactions. We report proteome-wide maps of protein-binding propensity for 407 structurally diverse small-molecule fragments. We verified that identified interactions can be advanced to active chemical probes of E3 ubiquitin ligases, transporters, and kinases. Integrating machine learning binary classifiers further enabled interpretable predictions of fragment behavior in cells. The resulting resource of fragment-protein interactions and predictive models will help to elucidate principles of molecular recognition and expedite ligand discovery efforts for hitherto undrugged proteins.

DOI: https://doi.org/10.1126/science.adk5864
Nat Microbiol. 2024 Apr 22.

Phage defence system CBASS is regulated by a prokaryotic E2 enzyme that imitates the ubiquitin pathway.

Yan Yan, Jun Xiao, Fengtao Huang, Wei Xian, Bingbing Yu, Rui Cheng, Hui Wu, Xueling Lu, Xionglue Wang, Wenjing Huang, Jing Li, Greater Kayode Oyejobi, Carol V Robinson, Hao Wu, Di Wu, Xiaoyun Liu, Longfei Wang, Bin Zhu.

The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, using biochemical, genetic, cryo-electron microscopy and mass spectrometry investigations, we discover that the E2 enzyme from Serratia marcescens regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.

DOI: https://doi.org/10.1038/s41564-024-01684-z
Plant Cell. 2024 Apr 24. pii: koae129. [Epub ahead of print]

Erasing marks: Functions of plant deubiquitylating enzymes in modulating the ubiquitin code.

Karin Vogel, Erika Isono.

Plant cells need to respond to environmental stimuli and developmental signals accurately and promptly. Ubiquitylation is a reversible posttranslational modification that enables the adaptation of cellular proteostasis to internal or external factors. The different topologies of ubiquitin linkages serve as the structural basis for the ubiquitin code, which can be interpreted by ubiquitin-binding proteins or readers in specific processes. The ubiquitylation status of target proteins is regulated by ubiquitylating enzymes or writers, and deubiquitylating enzymes (DUBs) or erasers. DUBs can remove ubiquitin molecules from target proteins. Arabidopsis (A. thaliana) DUBs belong to seven protein families and exhibit a wide range of functions and play an important role in regulating selective protein degradation processes, including proteasomal-, endocytic-, and autophagic protein degradation. DUBs also shape the epigenetic landscape and modulate DNA damage repair processes. In this review, we summarize the current knowledge on DUBs in plants, their cellular functions, and the regulatory mechanisms involved in the spatiotemporal regulation of plant DUBs.

DOI: https://doi.org/10.1093/plcell/koae129
Cell Rep. 2024 Apr 25. pii: S2211-1247(24)00480-7. [Epub ahead of print]43(5): 114152

Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1β production in human macrophages and microglia.

Zhu Liang, Andreas Damianou, Iolanda Vendrell, Edward Jenkins, Frederik H Lassen, Sam J Washer, Athina Grigoriou, Guihai Liu, Gangshun Yi, Hantao Lou, Fangyuan Cao, Xiaonan Zheng, Ricardo A Fernandes, Tao Dong, Edward W Tate, Elena Di Daniel, Benedikt M Kessler.

  Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labeling, affinity purification, and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-interleukin-1β (IL-1β) levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1β cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1β production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.

Keywords:  CP: Immunology; DUB; IL-1β; NLRP3; UCH-L1; deubiquitylase; inflammasome; macrophages; microglia; monocytes; protease inhibitor; ubiquitin; ubiquitin C-terminal hydrolase

DOI:  https://doi.org/10.1016/j.celrep.2024.114152
Autophagy. 2024 Apr 23. 1-12

The Wolfram-like variant WFS1E864K destabilizes MAM and compromises autophagy and mitophagy in human and mice.

Simone Patergnani, Méghane S Bataillard, Alberto Danese, Stacy Alves, Chantal Cazevieille, René Valéro, Lisbeth Tranebjærg, Tangui Maurice, Paolo Pinton, Benjamin Delprat, Elodie M Richard.

  Dominant variants in WFS1 (wolframin ER transmembrane glycoprotein), the gene coding for a mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) resident protein, have been associated with Wolfram-like syndrome (WLS). In vitro and in vivo, WFS1 loss results in reduced ER to mitochondria calcium (Ca2+) transfer, mitochondrial dysfunction, and enhanced macroautophagy/autophagy and mitophagy. However, in the WLS pathological context, whether the mutant protein triggers the same cellular processes is unknown. Here, we show that in human fibroblasts and murine neuronal cultures the WLS protein WFS1E864K leads to decreases in mitochondria bioenergetics and Ca2+ uptake, deregulation of the mitochondrial quality system mechanisms, and alteration of the autophagic flux. Moreover, in the Wfs1E864K mouse, these alterations are concomitant with a decrease of MAM number. These findings reveal pathophysiological similarities between WS and WLS, highlighting the importance of WFS1 for MAM's integrity and functionality. It may open new treatment perspectives for patients with WLS.Abbreviations: BafA1: bafilomycin A1; ER: endoplasmic reticulum; HSPA9/GRP75: heat shock protein family A (Hsp70) member 9; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; MAM: mitochondria-associated endoplasmic reticulum membrane; MCU: mitochondrial calcium uniporter; MFN2: mitofusin 2; OCR: oxygen consumption rate; ROS: reactive oxygen species; ROT/AA: rotenone+antimycin A; VDAC1: voltage dependent anion channel 1; WLS: Wolfram-like syndrome; WS: Wolfram syndrome; WT: wild-type.

Keywords:  Autophagy; WFS1; Wolfram-like syndrome; mitochondria-associated endoplasmic reticulum membrane; mitophagy

DOI:  https://doi.org/10.1080/15548627.2024.2341588
Nat Struct Mol Biol. 2024 Apr 22.

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation.

Julio C Y Liu, Leena Ackermann, Saskia Hoffmann, Zita Gál, Ivo A Hendriks, Charu Jain, Louise Morlot, Michael H Tatham, Gian-Luca McLelland, Ronald T Hay, Michael Lund Nielsen, Thijn Brummelkamp, Peter Haahr, Niels Mailand.

Protein SUMOylation provides a principal driving force for cellular stress responses, including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets, including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk.

DOI: https://doi.org/10.1038/s41594-024-01294-7
bioRxiv. 2024 Apr 21. pii: 2024.04.18.590103. [Epub ahead of print]

Nuclear speckle rejuvenation alleviates proteinopathies at the expense of YAP1.

William Dion, Yuren Tao, Maci Chambers, Shanshan Zhao, Riley K Arbuckle, Michelle Sun, Syeda Kubra, Yuhang Nie, Megan Ye, Mads B Larsen, Daniel Camarco, Eleanor Ickes, Claire DuPont, Haokun Wang, Bingjie Wang, Silvia Liu, Shaohua Pi, Bill B Chen, Yuanyuan Chen, Xu Chen, Bokai Zhu.

Current treatments targeting individual protein quality control have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common upstream druggable target capable of global proteostasis modulation. Building on our prior research establishing nuclear speckles as pivotal organelles responsible for global proteostasis transcriptional control, we aim to alleviate proteinopathies through nuclear speckle rejuvenation. We identified pyrvinium pamoate as a small-molecule nuclear speckle rejuvenator that enhances protein quality control while suppressing YAP1 signaling via decreasing the surface tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, pyrvinium pamoate reduced tauopathy and alleviated retina degeneration by promoting autophagy and ubiquitin-proteasome system. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity were also observed in human tauopathies. Our study uncovers novel therapeutic targets for tackling protein misfolding disorders within an expanded proteostasis framework encompassing nuclear speckles and YAP1.

DOI: https://doi.org/10.1101/2024.04.18.590103
Proc Natl Acad Sci U S A. 2024 Apr 30. 121(18): e2313107121

Maximum entropy determination of mammalian proteome dynamics.

Alexander J Dear, Gonzalo A Garcia, Georg Meisl, Galen A Collins, Tuomas P J Knowles, Alfred L Goldberg.

  Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

Keywords:  maximum entropy; protein degradation; protein half-lives; proteome; proteostasis

DOI:  https://doi.org/10.1073/pnas.2313107121
bioRxiv. 2024 Apr 19. pii: 2024.04.19.590252. [Epub ahead of print]

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR).

Stanley I Goldstein, Alice C Fan, Zihao Wang, Sai K Naineni, Johan Lengqvist, Alexey Chernobrovkin, Steve B Garcia-Gutierrez, Regina Cencic, Kesha Patel, Sidong Huang, Lauren E Brown, Andrew Emili, John A Porco.

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP and RNA (TSAR) as a template for proteome-wide discovery of substrate-dependent ligand binding. Using proteomic thermal shift assays, we show that simple biochemical additives can facilitate detection of target engagement in native cell lysates. We apply our approach to rocaglates, a family of molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) family of RNA helicases. To identify unexpected interactions, we optimized a target class-specific thermal denaturation window and evaluated ATP analog and RNA probe dependencies for key rocaglate-DDX interactions. We report novel DDX targets of the rocaglate clamping spectrum, confirm that DDX3X is a common target of several widely studied analogs, and provide structural insights into divergent DDX3X affinities between synthetic rocaglates. We independently validate novel targets of high-profile rocaglates, including the clinical candidate Zotatifin ( eFT226 ), using limited proteolysis-mass spectrometry and fluorescence polarization experiments. Taken together, our study provides a model for screening uncompetitive inhibitors using a systematic chemical-proteomics approach to uncover actionable DDX targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicase targets.

DOI: https://doi.org/10.1101/2024.04.19.590252
ACS Chem Neurosci. 2024 Apr 24.

Proteostatic Remodeling of Small Heat Shock Chaperones─Crystallins by Ran-Binding Protein 2─and the Peptidyl-Prolyl cis-trans Isomerase and Chaperone Activities of Its Cyclophilin Domain.

Hemangi Patil, Haiqing Yi, Kyoung-In Cho, Paulo A Ferreira.

  Disturbances in protein phase transitions promote protein aggregation─a neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also regulate phase transitions and proteostasis by suppressing protein aggregation. Ranbp2 haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against phototoxicity by proteostatic regulations of neuroprotective substrates of Ranbp2 and by suppressing the buildup of polyubiquitylated substrates. Losses of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 recapitulate molecular effects of Ranbp2 haploinsufficiency. These CY impairments also stimulate deubiquitylation activities and phase transitions of 19S cap subunits of the 26S proteasome that associates with Ranbp2. However, links between CY moonlighting activity, substrate ubiquitylation, and proteostasis remain incomplete. Here, we reveal the Ranbp2 regulation of small heat shock chaperones─crystallins in the chorioretina by proteomics of mice with total or selective modular deficits of Ranbp2. Specifically, loss of CY PPIase of Ranbp2 upregulates αA-Crystallin, which is repressed in adult nonlenticular tissues. Conversely, impairment of CY's chaperone activity opposite to the PPIase pocket downregulates a subset of αA-Crystallin's substrates, γ-crystallins. These CY-dependent effects cause age-dependent and chorioretinal-selective declines of ubiquitylated substrates without affecting the chorioretinal morphology. A model emerges whereby inhibition of Ranbp2's CY PPIase remodels crystallins' expressions, subdues molecular aging, and preordains the chorioretina to neuroprotection by augmenting the chaperone capacity and the degradation of polyubiquitylated substrates against proteostatic impairments. Further, the druggable Ranbp2 CY holds pan-therapeutic potential against proteotoxicity and neurodegeneration.

Keywords:  Ran-binding protein 2 (Ranbp2); chaperone; crystallins; cyclophilin; heat shock proteins; peptidyl-prolyl cis−trans isomerase (PPIase); proteostasis; proteotoxicity

DOI:  https://doi.org/10.1021/acschemneuro.3c00792
Nat Commun. 2024 Apr 26. 15(1): 3558

Mechanism of Ψ-Pro/C-degron recognition by the CRL2FEM1B ubiquitin ligase.

Xinyan Chen, Anat Raiff, Shanshan Li, Qiong Guo, Jiahai Zhang, Hualin Zhou, Richard T Timms, Xuebiao Yao, Stephen J Elledge, Itay Koren, Kaiming Zhang, Chao Xu.

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.

DOI: https://doi.org/10.1038/s41467-024-47890-5
Cell. 2024 Apr 16. pii: S0092-8674(24)00320-9. [Epub ahead of print]

Integrative spatial analysis reveals a multi-layered organization of glioblastoma.

Alissa C Greenwald, Noam Galili Darnell, Rouven Hoefflin, Dor Simkin, Christopher W Mount, L Nicolas Gonzalez Castro, Yotam Harnik, Sydney Dumont, Dana Hirsch, Masashi Nomura, Tom Talpir, Merav Kedmi, Inna Goliand, Gioele Medici, Julie Laffy, Baoguo Li, Vamsi Mangena, Hadas Keren-Shaul, Michael Weller, Yoseph Addadi, Marian C Neidert, Mario L Suvà, Itay Tirosh.

  Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.

Keywords:  glioblastoma; glioma; hypoxia; intratumor heterogeneity; spatial proteomics; spatial transcriptomics

DOI:  https://doi.org/10.1016/j.cell.2024.03.029
J Clin Invest. 2024 Apr 25. pii: e176355. [Epub ahead of print]

Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer.

Xiaochao Tan, Shike Wang, Guan-Yu Xiao, Chao Wu, Xin Liu, Biyao Zhou, Jiang Yu, Dzifa Yawa Duose, Yuanxin Xi, Jing Wang, Kunika Gupta, Apar Pataer, Jack A Roth, Michael P Kim, Fengju Chen, Chad J Creighton, William K Russell, Jonathan M Kurie.

  Cancer cells exhibit heightened secretory states that drive tumor progression. Here, we identify a chromosome 3q amplicon that serves as a platform for secretory regulation in cancer. The 3q amplicon encodes multiple Golgi-resident proteins, including the scaffold Golgi integral membrane protein 4 (GOLIM4) and the ion channel ATPase Secretory Pathway Ca2+ Transporting 1 (ATP2C1). We show that GOLIM4 recruits ATP2C1 and Golgi phosphoprotein 3 (GOLPH3) to coordinate calcium-dependent cargo loading and Golgi membrane bending and vesicle scission. GOLIM4 depletion disrupts the protein complex, resulting in a secretory blockade that inhibits the progression of 3q-amplified malignancies. In addition to its role as a scaffold, GOLIM4 maintains intracellular manganese (Mn) homeostasis by binding excess Mn in the Golgi lumen, which initiates the routing of Mn-bound GOLIM4 to lysosomes for degradation. We show that Mn treatment inhibits the progression of multiple types of 3q-amplified malignancies by degrading GOLIM4, resulting in a secretory blockade that interrupts pro-survival autocrine loops and attenuates pro-metastatic processes in the tumor microenvironment. Potentially underlying the selective activity of Mn against 3q-amplified malignancies, ATP2C1 co-amplification increases Mn influx into the Golgi lumen, resulting in a more rapid degradation of GOLIM4. These findings show that functional cooperativity between co-amplified genes underlies heightened secretion and a targetable secretory addiction in 3q-amplified malignancies.

Keywords:  Cancer gene therapy; Cell biology; Lung cancer; Oncology; Protein traffic

DOI:  https://doi.org/10.1172/JCI176355
Cell Rep. 2024 Apr 23. pii: S2211-1247(24)00462-5. [Epub ahead of print]43(5): 114134

The MYCN 5' UTR as a therapeutic target in neuroblastoma.

Marina P Volegova, Lauren E Brown, Ushashi Banerjee, Ruben Dries, Bandana Sharma, Alyssa Kennedy, John A Porco, Rani E George.

  Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

Keywords:  CP: Cancer; CP: Molecular biology; MYCN; MYCN amplification; PAR-CLIP; amidino-rocaglate; eIF4A1; neuroblastoma; ribosome profiling; rocaglate; translation; translation regulation

DOI:  https://doi.org/10.1016/j.celrep.2024.114134
bioRxiv. 2024 Apr 15. pii: 2024.04.15.589616. [Epub ahead of print]

Chemical codes promote selective compartmentalization of proteins.

Henry R Kilgore, Itamar Chinn, Peter G Mikhael, Ilan Mitnikov, Catherine Van Dongen, Guy Zylberberg, Lena Afeyan, Salman Banani, Susana Wilson-Hawken, Tong Ihn Lee, Regina Barzilay, Richard A Young.

Cells have evolved mechanisms to distribute ∼10 billion protein molecules to subcellular compartments where diverse proteins involved in shared functions must efficiently assemble. Such assembly is presumed to unfold as a result of specific interactions between biomolecules; however, recent evidence suggests that distinctive chemical environments within subcellular compartments may also play an important role. Here, we test the hypothesis that protein groups with shared functions also share codes that guide them to compartment destinations. To test our hypothesis, we developed a transformer large language model, called ProtGPS, that predicts with high performance the compartment localization of human proteins excluded from the training set. We then demonstrate ProtGPS can be used for guided generation of novel protein sequences that selectively assemble into specific compartments in cells. Furthermore, ProtGPS predictions were sensitive to disease-associated mutations that produce changes in protein compartmentalization, suggesting that this type of pathogenic dysfunction can be discovered in silico. Our results indicate that protein sequences contain not only a folding code, but also a previously unrecognized chemical code governing their distribution in specific cellular compartments.

DOI: https://doi.org/10.1101/2024.04.15.589616
Cancer Lett. 2024 Apr 24. pii: S0304-3835(24)00288-X. [Epub ahead of print] 216895

ANKZF1 knockdown inhibits glioblastoma progression by promoting intramitochondrial protein aggregation through mitoRQC.

Guangzhao Li, Zongqi Wang, Bixi Gao, Kun Dai, Xiaowang Niu, Xiang Li, Yunjiang Wang, Longyuan Li, Xin Wu, Haiying Li, Zhengquan Yu, Zhong Wang, Gang Chen.

  Protein homeostasis is fundamental to the development of tumors. Ribosome-associated quality-control (RQC) is able to add alanine and threonine to the stagnant polypeptide chain C-terminal (CAT-tail) when protein translation is hindered, while Ankyrin repeat and zinc-finger domain-containing-protein 1 (ANKZF1) can counteract the formation of the CAT-tail, preventing the aggregation of polypeptide chains. In particular, ANKZF1 plays an important role in maintaining mitochondrial protein homeostasis by mitochondrial RQC (mitoRQC) after translation stagnation of precursor proteins targeting mitochondria. However, the role of ANKZF1 in glioblastoma is unclear. Therefore, the current study was aimed to investigate the effects of ANKZF1 in glioblastoma cells and a nude mouse glioblastoma xenograft model. Here, we reported that knockdown of ANKZF1 in glioblastoma cells resulted in the accumulation of CAT-tail in mitochondria, leading to the activated mitochondrial unfolded protein response (UPRmt) and inhibits glioblastoma malignant progression. Excessive CAT-tail sequestered mitochondrial chaperones HSP60, mtHSP70 and proteases LONP1 as well as mitochondrial respiratory chain subunits ND1, Cytb, mtCO2 and ATP6, leading to mitochondrial oxidative phosphorylation dysfunction, membrane potential impairment, and mitochondrial apoptotic pathway activation. Our study highlights ANKZF1 as a valuable target for glioblastoma intervention and provides an innovative insight for the treatment of glioblastoma through the regulating of mitochondrial protein homeostasis.

Keywords:  Ankyrin repeat and zinc-finger domain-containing protein 1; CAT-tail; Glioblastoma; Mitochondrial Unfolded Protein Response; Protein aggresome; Ribosome related Quality Control

DOI:  https://doi.org/10.1016/j.canlet.2024.216895
bioRxiv. 2024 Apr 08. pii: 2024.04.04.588107. [Epub ahead of print]

STING promotes homeostatic maintenance of tissues and confers longevity with aging.

Jacob W Hopkins, Katherine B Sulka, Machlan Sawden, Kimberly A Carroll, Ronald D Brown, Stephen C Bunnell, Alexander Poltorak, Albert Tai, Eric R Reed, Shruti Sharma.

Local immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in various in vitro and in vivo models of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immune fingerprint for each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING- sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues. Ex vivo analyses reveal that basal STING- signaling limits the susceptibility of TRMs to death-inducing stimuli and determines their in situ localization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging.
HIGHLIGHTS: Aging tissues are associated with tissue-autonomous immune fingerprints, primarily driven by interactions of tissue stromal and myeloid populations. STING shapes these immune fingerprints of aging tissues in unexpected ways.Loss of STING alters the location, numbers, and viability of tissue resident macrophages.STING signaling is critical for longer lifespans and maintenance of tissue architecture.

DOI: https://doi.org/10.1101/2024.04.04.588107
Nat Chem Biol. 2024 Apr 22.

UFL1 triggers replication fork degradation by MRE11 in BRCA1/2-deficient cells.

Tian Tian, Junliang Chen, Huacun Zhao, Yulin Li, Feiyu Xia, Jun Huang, Jinhua Han, Ting Liu.

The stabilization of stalled forks has emerged as a crucial mechanism driving resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient tumors. Here, we identify UFL1, a UFM1-specific E3 ligase, as a pivotal regulator of fork stability and the response to PARP inhibitors in BRCA1/2-deficient cells. On replication stress, UFL1 localizes to stalled forks and catalyzes the UFMylation of PTIP, a component of the MLL3/4 methyltransferase complex, specifically at lysine 148. This modification facilitates the assembly of the PTIP-MLL3/4 complex, resulting in the enrichment of H3K4me1 and H3K4me3 at stalled forks and subsequent recruitment of the MRE11 nuclease. Consequently, loss of UFL1, disruption of PTIP UFMylation or overexpression of the UFM1 protease UFSP2 protects nascent DNA strands from extensive degradation and confers resistance to PARP inhibitors in BRCA1/2-deficient cells. These findings provide mechanistic insights into the processes underlying fork instability in BRCA1/2-deficient cells and offer potential therapeutic avenues for the treatment of BRCA1/2-deficient tumors.

DOI: https://doi.org/10.1038/s41589-024-01611-7
Nat Commun. 2024 Apr 24. 15(1): 3302

TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Ishier Raote, Ann-Helen Rosendahl, Hanna-Maria Häkkinen, Carina Vibe, Ismail Küçükaylak, Mugdha Sawant, Lena Keufgens, Pia Frommelt, Kai Halwas, Katrina Broadbent, Marina Cunquero, Gustavo Castro, Marie Villemeur, Julian Nüchel, Anna Bornikoel, Binita Dam, Ravindra K Zirmire, Ravi Kiran, Carlo Carolis, Jordi Andilla, Pablo Loza-Alvarez, Verena Ruprecht, Colin Jamora, Felix Campelo, Marcus Krüger, Matthias Hammerschmidt, Beate Eckes, Ines Neundorf, Thomas Krieg, Vivek Malhotra.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

DOI: https://doi.org/10.1038/s41467-024-47004-1
bioRxiv. 2024 Apr 12. pii: 2024.04.09.588596. [Epub ahead of print]

Predictomes: A classifier-curated database of AlphaFold-modeled protein-protein interactions.

Ernst W Schmid, Johannes C Walter.

Protein-protein interactions (PPIs) are ubiquitous in biology, yet a comprehensive structural characterization of the PPIs underlying biochemical processes is lacking. Although AlphaFold-Multimer (AF-M) has the potential to fill this knowledge gap, standard AF-M confidence metrics do not reliably separate relevant PPIs from an abundance of false positive predictions. To address this limitation, we used machine learning on well curated datasets to train a S tructure P rediction and O mics informed C lassifier called SPOC that shows excellent performance in separating true and false PPIs, including in proteome-wide screens. We applied SPOC to an all-by-all matrix of nearly 300 human genome maintenance proteins, generating ∼40,000 predictions that can be viewed at predictomes.org, where users can also score their own predictions with SPOC. High confidence PPIs discovered using our approach suggest novel hypotheses in genome maintenance. Our results provide a framework for interpreting large scale AF-M screens and help lay the foundation for a proteome-wide structural interactome.

DOI: https://doi.org/10.1101/2024.04.09.588596
Biochim Biophys Acta Mol Basis Dis. 2024 Apr 20. pii: S0925-4439(24)00182-0. [Epub ahead of print]1870(5): 167193

The IRE1α-XBP1 arm of the unfolded protein response is a host factor activated in SARS-CoV-2 infection.

Jose Javier Fernández, Arturo Marín, Romel Rosales, Rebekah Penrice-Randal, Petra Mlcochova, Yolanda Alvarez, Fernando Villalón-Letelier, Soner Yildiz, Enrique Pérez, Raveen Rathnasinghe, Anastasija Cupic, Thomas Kehrer, Melissa B Uccellini, Sara Alonso, Fernando Martínez, Briana Lynn McGovern, Jordan J Clark, Parul Sharma, Yolanda Bayón, Andrés Alonso, Randy A Albrecht, Kris M White, Michael Schotsaert, Lisa Miorin, James P Stewart, Julian A Hiscox, Ravindra K Gupta, Nerea Irigoyen, Adolfo García-Sastre, Mariano Sánchez Crespo, Nieves Fernández.

  SARS-CoV-2 infection can cause severe pneumonia, wherein exacerbated inflammation plays a major role. This is reminiscent of the process commonly termed cytokine storm, a condition dependent on a disproportionated production of cytokines. This state involves the activation of the innate immune response by viral patterns and coincides with the biosynthesis of the biomass required for viral replication, which may overwhelm the capacity of the endoplasmic reticulum and drive the unfolded protein response (UPR). The UPR is a signal transduction pathway composed of three branches that is initiated by a set of sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These sensors control adaptive processes, including the transcriptional regulation of proinflammatory cytokines. Based on this background, the role of the UPR in SARS-CoV-2 replication and the ensuing inflammatory response was investigated using in vivo and in vitro models of infection. Mice and Syrian hamsters infected with SARS-CoV-2 showed a sole activation of the Ire1α-Xbp1 arm of the UPR associated with a robust production of proinflammatory cytokines. Human lung epithelial cells showed the dependence of viral replication on the expression of UPR-target proteins branching on the IRE1α-XBP1 arm and to a lower extent on the PERK route. Likewise, activation of the IRE1α-XBP1 branch by Spike (S) proteins from different variants of concern was a uniform finding. These results show that the IRE1α-XBP1 system enhances viral replication and cytokine expression and may represent a potential therapeutic target in SARS-CoV-2 severe pneumonia.

Keywords:  COVID-19; Cytokines; Fluvoxamine; Pneumonia; TLR; Transcription factors; Unfolded protein response; Variants of concern; Viral sepsis

DOI:  https://doi.org/10.1016/j.bbadis.2024.167193
Proc Natl Acad Sci U S A. 2024 Apr 30. 121(18): e2318619121

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

David J Sherman, Lei Liu, Jennifer L Mamrosh, Jiansong Xie, John Ferbas, Brett Lomenick, Mark S Ladinsky, Rati Verma, Ingrid C Rulifson, Raymond J Deshaies.

  Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Keywords:  Golgi apparatus; PNPLA3; fatty liver disease; lipid droplet; phosphoinositide

DOI:  https://doi.org/10.1073/pnas.2318619121
Nat Chem Biol. 2024 Apr 24.

Molecular recording of calcium signals via calcium-dependent proximity labeling.

J Wren Kim, Adeline J H Yong, Erin E Aisenberg, Joseph H Lobel, Wei Wang, Ted M Dawson, Valina L Dawson, Ruixuan Gao, Yuh Nung Jan, Helen S Bateup, Nicholas T Ingolia.

Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, calcium transients play a central role in regulating neurotransmitter release and synaptic plasticity. However, it is challenging to capture the molecular events associated with these localized and ephemeral calcium signals. Here we present an engineered biotin ligase that generates permanent molecular traces in a calcium-dependent manner. The enzyme, calcium-dependent BioID (Cal-ID), biotinylates nearby proteins within minutes in response to elevated local calcium levels. The biotinylated proteins can be identified via mass spectrometry and visualized using microscopy. In neurons, Cal-ID labeling is triggered by neuronal activity, leading to prominent protein biotinylation that enables transcription-independent activity labeling in the brain. In summary, Cal-ID produces a biochemical record of calcium signals and neuronal activity with high spatial resolution and molecular specificity.

DOI: https://doi.org/10.1038/s41589-024-01603-7
Proc Natl Acad Sci U S A. 2024 Apr 30. 121(18): e2322520121

PARP1 UFMylation ensures the stability of stalled replication forks.

Yamin Gong, Zhifeng Wang, Wen Zong, Ruifeng Shi, Wenli Sun, Sijia Wang, Bin Peng, Shunichi Takeda, Zhao-Qi Wang, Xingzhi Xu.

  The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.

Keywords:  DNA replication; PARP1; UFMylation; replication stress

DOI:  https://doi.org/10.1073/pnas.2322520121
J Cell Biol. 2024 Aug 05. pii: e202308049. [Epub ahead of print]223(8):

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

Benjamin P Robinson, Naomi R Bass, Priyanka Bhakt, Elias T Spiliotis.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

DOI: https://doi.org/10.1083/jcb.202308049
PLoS One. 2024 ;19(4): e0297122

AviTrap: A novel solution to achieve complete biotinylation.

Cheng Lu, Jack Bevers, Tulika Tyagi, Hao To, May Lin, Shu Ti, Gerry Nakamura, WeiYu Lin, Yongmei Chen, Yan Wu, Hong Li, Jiansheng Wu, Feng Wang.

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.

DOI: https://doi.org/10.1371/journal.pone.0297122
Nat Commun. 2024 Apr 24. 15(1): 3467

The phosphorylation of carboxyl-terminal eIF2α by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis.

Hui-Hsien Chang, Lin-Chen Huang, Karen S Browning, Enamul Huq, Mei-Chun Cheng.

Light triggers an enhancement of global translation during photomorphogenesis in Arabidopsis, but little is known about the underlying mechanisms. The phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) at a conserved serine residue in the N-terminus has been shown as an important mechanism for the regulation of protein synthesis in mammalian and yeast cells. However, whether the phosphorylation of this residue in plant eIF2α plays a role in regulation of translation remains elusive. Here, we show that the quadruple mutant of SUPPRESSOR OF PHYA-105 family members (SPA1-SPA4) display repressed translation efficiency after light illumination. Moreover, SPA1 directly phosphorylates the eIF2α C-terminus under light conditions. The C-term-phosphorylated eIF2α promotes translation efficiency and photomorphogenesis, whereas the C-term-unphosphorylated eIF2α results in a decreased translation efficiency. We also demonstrate that the phosphorylated eIF2α enhances ternary complex assembly by promoting its affinity to eIF2β and eIF2γ. This study reveals a unique mechanism by which light promotes translation via SPA1-mediated phosphorylation of the C-terminus of eIF2α in plants.

DOI: https://doi.org/10.1038/s41467-024-47848-7