bims-proteo Biomed News
on Proteostasis
Issue of 2024–09–22
forty-six papers selected by
Eric Chevet, INSERM
  1. Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.
  2. Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination.
  3. Non-cell-autonomous regulation of germline proteostasis by insulin/IGF-1 signaling-induced dietary peptide uptake via PEPT-1.
  4. ER-phagy restrains inflammatory responses through its receptor UBAC2.
  5. O-GlcNAc modification of HSP27 alters its protein interactions and promotes refolding of proteins through the BAG3/HSP70 co-chaperone.
  6. Met1-linked ubiquitination in cell signaling regulation.
  7. Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes.
  8. Endoplasmic Reticulum Stress and Obesity.
  9. Targeting cancer with small-molecule pan-KRAS degraders.
  10. Systems mapping of bidirectional endosomal transport through the crowded cell.
  11. Translation elongation inhibitors stabilize select short-lived transcripts.
  12. The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1.
  13. The partitioning of fatty acids between membrane and storage lipids controls ER membrane expansion.
  14. Activation of parkin by a molecular glue.
  15. Co-opting templated aggregation to degrade pathogenic tau assemblies and improve motor function.
  16. Structural basis for translational control by the human 48S initiation complex.
  17. Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis.
  18. USP37 prevents premature disassembly of stressed replisomes by TRAIP.
  19. HSPB6: A lipid-dependent molecular chaperone inhibits α-synuclein aggregation.
  20. Non-autophagic Golgi-LC3 lipidation facilitates TFE3 stress response against Golgi dysfunction.
  21. DNL343 is an investigational CNS penetrant eukaryotic initiation factor 2B activator that prevents and reverses the effects of neurodegeneration caused by the integrated stress response.
  22. Protein interactions in human pathogens revealed through deep learning.
  23. Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts.
  24. N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation.
  25. Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation.
  26. Protein UFMylation regulates early events during ribosomal DNA-damage response.
  27. The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation.
  28. TBK1 adaptor AZI2/NAP1 regulates NDP52-driven mitochondrial autophagy.
  29. Multi-omics analysis using antibody-based in situ biotinylation technique suggests the mechanism of Cajal body formation.
  30. Structural basis of human 20S proteasome biogenesis.
  31. Live-cell visualization of tau aggregation in human neurons.
  32. Proximity labeling defines the phagosome lumen proteome of murine and primary human macrophages.
  33. Feedback control of the heat shock response by spatiotemporal regulation of Hsp70.
  34. Targeted protein relocalization via protein transport coupling.
  35. Macroautophagy/autophagy promotes resistance to KRASG12D-targeted therapy through glutathione synthesis.
  36. Genome-Wide Mapping of RNA-Protein Associations via Sequencing.
  37. ER-plasma membrane contact sites deliver ER lipids and proteins for rapid cell surface expansion.
  38. Large library docking identifies positive allosteric modulators of the calcium-sensing receptor.
  39. Elevated VCP ATPase Activity Correlates With Disease Onset in Multisystem Proteinopathy-1.
  40. A tRNA modification with aminovaleramide facilitates AUA decoding in protein synthesis.
  41. Regulation of synapse density by Pumilio RNA-binding proteins.
  42. Spatial and signaling overlap of growth factor receptor systems at clathrin-coated sites.
  43. Plasma membrane curvature regulates the formation of contacts with the endoplasmic reticulum.
  44. Protocol for detecting glycoRNAs using metabolic labeling and northwestern blot.
  45. Migfilin promotes autophagic flux through direct interaction with SNAP29 and Vamp8.
  46. PPI-hotspotID for detecting protein-protein interaction hot spots from the free protein structure.
  1. bioRxiv. 2024 Sep 03. pii: 2024.08.30.610524. [Epub ahead of print]
    Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.
    Kelsey L Hickey, Alexandra Panov, Enya Miguel Whelan, Tillman Schäfer, Arda Mizrak, Ron R Kopito, Wolfgang Baumeister, Rubén Fernández-Busnadiego, J Wade Harper.
      A hallmark of neurodegenerative diseases is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription, and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.
    Keywords:  Biological Sciences; Cell Biology; NRF1; Protein aggregates; Protein quality control; Protein turnover; UBE3C; Ubiquitin-proteasome system
    DOI:  https://doi.org/10.1101/2024.08.30.610524
  2. bioRxiv. 2024 Sep 02. pii: 2024.09.02.610852. [Epub ahead of print]
    Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination.
    Zara Bukhari, Li Gu, Anneroos E Nederstigt, Logan J Cope, Derek L Bolhuis, Kim Harvey, Tristan Allen, Spencer Hill, Yujie Yang, Guy Lawson, Cai Lu, Tommy Tran, Leah Pineda, Leanne Low, Andrew Chiang, Jason Song, Michelle V Fong, Vanessa M Rangel, William K Chan, Gary Kleiger, Dennis Goldfarb, Craig A Vierra, Nicholas G Brown, Joseph S Harrison.
      Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC 50 to cisplatin and whole-cell proteomics reveal an increased abundance of ∼20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2D's central role in proteome management.
    DOI:  https://doi.org/10.1101/2024.09.02.610852
  3. EMBO J. 2024 Sep 16.
    Non-cell-autonomous regulation of germline proteostasis by insulin/IGF-1 signaling-induced dietary peptide uptake via PEPT-1.
    Tahir Muhammad, Stacey L Edwards, Allison C Morphis, Mary V Johnson, Vitor De Oliveira, Tomasz Chamera, Siyan Liu, Ngoc Gia Tuong Nguyen, Jian Li.
      Gametogenesis involves active protein synthesis and is proposed to rely on proteostasis. Our previous work in C. elegans indicates that germline development requires coordinated activities of insulin/IGF-1 signaling (IIS) and HSF-1, the central regulator of the heat shock response. However, the downstream mechanisms were not identified. Here, we show that depletion of HSF-1 from germ cells impairs chaperone gene expression, causing protein degradation and aggregation and, consequently, reduced fecundity and gamete quality. Conversely, reduced IIS confers germ cell resilience to HSF-1 depletion-induced protein folding defects and various proteotoxic stresses. Surprisingly, this effect was not mediated by an enhanced stress response, which underlies longevity in low IIS conditions, but by reduced ribosome biogenesis and translation rate. We found that IIS activates the expression of intestinal peptide transporter PEPT-1 by alleviating its repression by FOXO/DAF-16, allowing dietary proteins to be efficiently incorporated into an amino acid pool that fuels germline protein synthesis. Our data suggest this non-cell-autonomous pathway is critical for proteostasis regulation during gametogenesis.
    Keywords:  Germline Proteostasis; Heat Shock Factor 1; Insulin and IGF-1 Signaling; Peptide Uptake; Protein Synthesis
    DOI:  https://doi.org/10.1038/s44318-024-00234-x
  4. EMBO J. 2024 Sep 16.
    ER-phagy restrains inflammatory responses through its receptor UBAC2.
    Xing He, Haowei He, Zitong Hou, Zheyu Wang, Qinglin Shi, Tao Zhou, Yaoxing Wu, Yunfei Qin, Jun Wang, Zhe Cai, Jun Cui, Shouheng Jin.
      ER-phagy, a selective form of autophagic degradation of endoplasmic reticulum (ER) fragments, plays an essential role in governing ER homeostasis. Dysregulation of ER-phagy is associated with the unfolded protein response (UPR), which is a major clue for evoking inflammatory diseases. However, the molecular mechanism underpinning the connection between ER-phagy and disease remains poorly defined. Here, we identified ubiquitin-associated domain-containing protein 2 (UBAC2) as a receptor for ER-phagy, while at the same time being a negative regulator of inflammatory responses. UBAC2 harbors a canonical LC3-interacting region (LIR) in its cytoplasmic domain, which binds to autophagosomal GABARAP. Upon ER-stress or autophagy activation, microtubule affinity-regulating kinase 2 (MARK2) phosphorylates UBAC2 at serine (S) 223, promoting its dimerization. Dimerized UBAC2 interacts more strongly with GABARAP, thus facilitating selective degradation of the ER. Moreover, by affecting ER-phagy, UBAC2 restrains inflammatory responses and acute ulcerative colitis (UC) in mice. Our findings indicate that ER-phagy directed by a MARK2-UBAC2 axis may provide targets for the treatment of inflammatory disease.
    Keywords:  Colitis; ER-phagy; Inflammatory Responses; MARK2; UBAC2
    DOI:  https://doi.org/10.1038/s44318-024-00232-z
  5. Protein Sci. 2024 Oct;33(10): e5173
    O-GlcNAc modification of HSP27 alters its protein interactions and promotes refolding of proteins through the BAG3/HSP70 co-chaperone.
    Afraah Javed, Oleta T Johnson, Aaron T Balana, Regan F Volk, Andreas Langen, Benjamin S Ahn, Balyn W Zaro, Jason E Gestwicki, Matthew R Pratt.
      Almost all types of cellular stress induce post-translational O-GlcNAc modifications of proteins, and this increase promotes cell survival. We previously demonstrated that O-GlcNAc on certain small heat shock proteins (sHSPs), including HSP27, directly increases their chaperone activity as one potential protective mechanism. Here, we furthered our use of synthetic proteins to prepare biotinylated sHSPs and show that O-GlcNAc modification of HSP27 also changes how it interacts within the sHSP system and the broader HSP network. Specifically, we show that O-GlcNAc modified HSP27 binds more strongly to the co-chaperone protein BAG3, which then promotes refolding of a model substrate by HSP70. We use proteomics to identify other potential HSP27 interactions that are changed by O-GlcNAc, including one that we confirm with another sHSP, αB-crystallin. These findings add additional evidence for O-GlcNAc as a switch for regulating protein-protein interactions and for modifications of chaperones as one mechanism by which O-GlcNAc protects against protein aggregation.
    Keywords:  O‐GlcNAc; aggregation; heat shock protein; refolding
    DOI:  https://doi.org/10.1002/pro.5173
  6. Biophys Rep. 2024 Aug 31. 10(4): 230-240
    Met1-linked ubiquitination in cell signaling regulation.
    Yanmin Guo, Yuqin Zhao, Yu-Sheng Cong.
      Met1-linked ubiquitination (Met1-Ub), also known as linear ubiquitination, is a newly identified atypical type of polyubiquitination that is assembled via the N-terminal methionine (Met1) rather than an internal lysine (Lys) residue of ubiquitin. The linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L and SHARPIN is the sole E3 ubiquitin ligase that specifically generates Met1-linked ubiquitin chains. The physiological role of LUBAC-mediated Met1-Ub has been first described as activating NF-κB signaling through the Met1-Ub modification of NEMO. However, accumulating evidence shows that Met1-Ub is broadly involved in other cellular pathways including MAPK, Wnt/β-Catenin, PI3K/AKT and interferon signaling, and participates in various cellular processes including angiogenesis, protein quality control and autophagy, suggesting that Met1-Ub harbors a potent signaling capacity. Here, we review the formation and cellular functions of Met1-linked ubiquitin chains, with an emphasis on the recent advances in the cellular mechanisms by which Met1-Ub controls signaling transduction.
    Keywords:  Cell signaling; LUBAC; Met1-linked ubiquitination; Ubiquitin
    DOI:  https://doi.org/10.52601/bpr.2024.230030
  7. Commun Biol. 2024 Sep 19. 7(1): 1179
    Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes.
    Jonathan D Taylor, Nathalie Barrett, Sergio Martinez Cuesta, Katelyn Cassidy, Fiona Pachl, James Dodgson, Radhika Patel, Tuula M Eriksson, Aidan Riley, Matthew Burrell, Christin Bauer, D Gareth Rees, Raffaello Cimbro, Andrew X Zhang, Ralph R Minter, James Hunt, Sandrine Legg.
      Proteins can be targeted for degradation by engineering biomolecules that direct them to the eukaryotic ubiquitination machinery. For instance, the fusion of an E3 ubiquitin ligase to a suitable target binding domain creates a 'biological Proteolysis-Targeting Chimera' (bioPROTAC). Here we employ an analogous approach where the target protein is recruited directly to a human E2 ubiquitin-conjugating enzyme via an attached target binding domain. Through rational design and screening we develop E2 bioPROTACs that induce the degradation of the human intracellular proteins SHP2 and KRAS. Using global proteomics, we characterise the target-specific and wider effects of E2 vs. VHL-based fusions. Taking SHP2 as a model target, we also employ a route to bioPROTAC discovery based on protein display libraries, yielding a degrader with comparatively weak affinity capable of suppressing SHP2-mediated signalling.
    DOI:  https://doi.org/10.1038/s42003-024-06803-4
  8. Adv Exp Med Biol. 2024 ;1460 373-390
    Endoplasmic Reticulum Stress and Obesity.
    Erkan Yilmaz.
      In recent years, the world has seen an alarming increase in obesity and is closely associated with insulin resistance, which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) plays in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably many causes for obesity-related insulin resistance and inflammation. One of the faulty mechanisms is protein homeostasis, protein quality control system included protein folding, chaperone activity, and ER-associated degradation leading to endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens, or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.
    Keywords:  Autophagy; Endoplasmic reticulum stress; Lipotoxicity; Obesity; Type 2 diabetes
    DOI:  https://doi.org/10.1007/978-3-031-63657-8_13
  9. Science. 2024 Sep 20. 385(6715): 1338-1347
    Targeting cancer with small-molecule pan-KRAS degraders.
    Johannes Popow, William Farnaby, Andreas Gollner, Christiane Kofink, Gerhard Fischer, Melanie Wurm, David Zollman, Andre Wijaya, Nikolai Mischerikow, Carina Hasenoehrl, Polina Prokofeva, Heribert Arnhof, Silvia Arce-Solano, Sammy Bell, Georg Boeck, Emelyne Diers, Aileen B Frost, Jake Goodwin-Tindall, Jale Karolyi-Oezguer, Shakil Khan, Theresa Klawatsch, Manfred Koegl, Roland Kousek, Barbara Kratochvil, Katrin Kropatsch, Arnel A Lauber, Ross McLennan, Sabine Olt, Daniel Peter, Oliver Petermann, Vanessa Roessler, Peggy Stolt-Bergner, Patrick Strack, Eva Strauss, Nicole Trainor, Vesna Vetma, Claire Whitworth, Siying Zhong, Jens Quant, Harald Weinstabl, Bernhard Kuster, Peter Ettmayer, Alessio Ciulli.
      Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.
    DOI:  https://doi.org/10.1126/science.adm8684
  10. Curr Biol. 2024 Sep 09. pii: S0960-9822(24)01138-2. [Epub ahead of print]
    Systems mapping of bidirectional endosomal transport through the crowded cell.
    Marlieke L M Jongsma, Nina Bakker, Lenard M Voortman, Roman I Koning, Erik Bos, Jimmy J L L Akkermans, Lennert Janssen, Jacques Neefjes.
      Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors.
    Keywords:  FYCO1; Lis1; Protrudin; RILP; Rab7; bidirectional transport; endosomes; membrane contact sites; motor proteins; optogenetics
    DOI:  https://doi.org/10.1016/j.cub.2024.08.026
  11. RNA. 2024 Sep 18. pii: rna.080138.124. [Epub ahead of print]
    Translation elongation inhibitors stabilize select short-lived transcripts.
    Nicolle A Rosa-Mercado, Allen R Buskirk, Rachel Green.
      Translation elongation inhibitors are commonly used to study different cellular processes. Yet, their specific impact on transcription and mRNA decay has not been thoroughly assessed. Here we use TimeLapse sequencing to investigate how translational stress impacts mRNA dynamics in human cells. Our results reveal that a distinct group of transcripts is stabilized in response to the translation elongation inhibitor emetine. These stabilized mRNAs are short-lived at steady state and many of them encode C2H2 zinc finger proteins. The codon usage of these stabilized transcripts is suboptimal compared to other expressed transcripts, including other short-lived mRNAs that are not stabilized after emetine treatment. Finally, we show that stabilization of these transcripts is independent of ribosome quality control factors and signaling pathways activated by ribosome collisions. Our data describe a group of short-lived transcripts whose degradation is particularly sensitive to the inhibition of translation elongation.
    Keywords:  codon optimality; mRNA stability; ribosome collisions; translation elongation
    DOI:  https://doi.org/10.1261/rna.080138.124
  12. Proc Natl Acad Sci U S A. 2024 Sep 24. 121(39): e2400531121
    The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1.
    Xiying Wang, Ji Geng, Suman Rimal, Yuxiu Sui, Jie Pan, Zhenghong Qin, Bingwei Lu.
      It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases.
    Keywords:  DRAM1; ER; ER-phagy; calcium homeostasis; lysosome
    DOI:  https://doi.org/10.1073/pnas.2400531121
  13. bioRxiv. 2024 Sep 05. pii: 2024.09.05.611378. [Epub ahead of print]
    The partitioning of fatty acids between membrane and storage lipids controls ER membrane expansion.
    Pawel K Lysyganicz, Antonio D Barbosa, Shoily Khondker, Nicholas A Stewart, George M Carman, Phillip J Stansfeld, Marcus K Dymond, Symeon Siniossoglou.
      The biogenesis of membrane-bound organelles involves the synthesis, remodelling and degradation of their constituent phospholipids. How these pathways regulate organelle size, remains still poorly understood. Here we demonstrate that a lipid degradation pathway inhibits the expansion of the endoplasmic reticulum (ER) membrane. Phospholipid diacylglycerol acyltransferases (PDATs) use endogenous phospholipids as fatty acyl donors to generate triglyceride stored in lipid droplets. The significance of this non-canonical triglyceride biosynthetic pathway has remained elusive. We find that the activity of the yeast PDAT Lro1 is regulated by a membrane- proximal domain facing the luminal side of the ER bilayer. To reveal the biological roles of PDATs, we engineered an Lro1 variant with derepressed activity. We show that active Lro1 mediates the retraction of ER membrane expansion driven by phospholipid synthesis. Furthermore, the subcellular distribution and membrane turnover activity of Lro1 are controlled by diacylglycerol, produced by the activity of Pah1, a conserved member of the lipin family. Collectively, our findings reveal a lipid metabolic network that regulates endoplasmic reticulum biogenesis by converting phospholipids into storage lipids.
    DOI:  https://doi.org/10.1101/2024.09.05.611378
  14. Nat Commun. 2024 Sep 19. 15(1): 7707
    Activation of parkin by a molecular glue.
    Véronique Sauvé, Eric Stefan, Nathalie Croteau, Thomas Goiran, Rayan Fakih, Nupur Bansal, Adelajda Hadzipasic, Jing Fang, Paramasivam Murugan, Shimin Chen, Edward A Fon, Warren D Hirst, Laura F Silvian, Jean-François Trempe, Kalle Gehring.
      Mutations in parkin and PINK1 cause early-onset Parkinson's disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson's disease.
    DOI:  https://doi.org/10.1038/s41467-024-51889-3
  15. Cell. 2024 Sep 10. pii: S0092-8674(24)00912-7. [Epub ahead of print]
    Co-opting templated aggregation to degrade pathogenic tau assemblies and improve motor function.
    Lauren V C Miller, Guido Papa, Marina Vaysburd, Shi Cheng, Paul W Sweeney, Annabel Smith, Catarina Franco, Taxiarchis Katsinelos, Melissa Huang, Sophie A I Sanford, Jonathan Benn, Jasmine Farnsworth, Katie Higginson, Holly Joyner, William A McEwan, Leo C James.
      Protein aggregation causes a wide range of neurodegenerative diseases. Targeting and removing aggregates, but not the functional protein, is a considerable therapeutic challenge. Here, we describe a therapeutic strategy called "RING-Bait," which employs an aggregating protein sequence combined with an E3 ubiquitin ligase. RING-Bait is recruited into aggregates, whereupon clustering dimerizes the RING domain and activates its E3 function, resulting in the degradation of the aggregate complex. We exemplify this concept by demonstrating the specific degradation of tau aggregates while sparing soluble tau. Unlike immunotherapy, RING-Bait is effective against both seeded and cell-autonomous aggregation. RING-Bait removed tau aggregates seeded from Alzheimer's disease (AD) and progressive supranuclear palsy (PSP) brain extracts and was also effective in primary neurons. We used a brain-penetrant adeno-associated virus (AAV) to treat P301S tau transgenic mice, reducing tau pathology and improving motor function. A RING-Bait strategy could be applied to other neurodegenerative proteinopathies by replacing the Bait sequence to match the target aggregate.
    Keywords:  Alzheimer’s disease; TRIM21; antibodies; gene therapy; neurobiology; neurodegeneration; protein engineering; targeted protein degradation; tauopathy; ubiquitination
    DOI:  https://doi.org/10.1016/j.cell.2024.08.024
  16. Nat Struct Mol Biol. 2024 Sep 17.
    Structural basis for translational control by the human 48S initiation complex.
    Valentyn Petrychenko, Sung-Hui Yi, David Liedtke, Bee-Zen Peng, Marina V Rodnina, Niels Fischer.
      The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome.
    DOI:  https://doi.org/10.1038/s41594-024-01378-4
  17. Autophagy. 2024 Sep 18.
    Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis.
    Rukmini Mukherjee, Anshu Bhattacharya, João Mello-Vieira, Santosh Kumar Kuncha, Marina Hoffmann, Alexis Gonzalez, Rajeshwari Rathore, Attinder Chadha, Donghyuk Shin, Thomas Colby, Ivan Matic, Shaeri Mukherjee, Mohit Misra, Ivan Dikic.
      The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner.
    Keywords:  Antioxidants; KEAP1; bacterial infection; legionella pneumophila; oxidative stress; reactive oxygen species
    DOI:  https://doi.org/10.1080/15548627.2024.2404375
  18. bioRxiv. 2024 Sep 04. pii: 2024.09.03.611025. [Epub ahead of print]
    USP37 prevents premature disassembly of stressed replisomes by TRAIP.
    Olga V Kochenova, Giuseppina D'Alessandro, Domenic Pilger, Ernst Schmid, Sean L Richards, Marcos Rios Garcia, Satpal S Jhujh, Andrea Voigt, Vipul Gupta, Christopher J Carnie, R Alex Wu, Nadia Gueorguieva, Grant S Stewart, Johannes C Walter, Stephen P Jackson.
      The E3 ubiquitin ligase TRAIP associates with the replisome and helps this molecular machine deal with replication stress. Thus, TRAIP promotes DNA inter-strand crosslink repair by triggering the disassembly of CDC45-MCM2-7-GINS (CMG) helicases that have converged on these lesions. However, disassembly of single CMGs that have stalled temporarily would be deleterious, suggesting that TRAIP must be carefully regulated. Here, we demonstrate that human cells lacking the de-ubiquitylating enzyme USP37 are hypersensitive to topoisomerase poisons and other replication stress-inducing agents. We further show that TRAIP loss rescues the hypersensitivity of USP37 knockout cells to topoisomerase inhibitors. In Xenopus egg extracts depleted of USP37, TRAIP promotes premature CMG ubiquitylation and disassembly when converging replisomes stall. Finally, guided by AlphaFold-Multimer, we discovered that binding to CDC45 mediates USP37's response to topological stress. In conclusion, we propose that USP37 protects genome stability by preventing TRAIP-dependent CMG unloading when replication stress impedes timely termination.
    DOI:  https://doi.org/10.1101/2024.09.03.611025
  19. iScience. 2024 Sep 20. 27(9): 110657
    HSPB6: A lipid-dependent molecular chaperone inhibits α-synuclein aggregation.
    Valentina Secco, Tatiana Tiago, Roxine Staats, Swapan Preet, Sean Chia, Michele Vendruscolo, Serena Carra.
      The process of protein misfolding and aggregation is associated with various cytotoxic effects. Understanding how this phenomenon is regulated by the protein homeostasis system, however, is difficult, since it takes place through a complex non-linear network of coupled microscopic steps, including primary nucleation, fibril elongation, and secondary nucleation, which depend on environmental factors. To address this problem, we studied how the aggregation of α-synuclein, a protein associated with Parkinson's disease, is modulated by molecular chaperones and lipid membranes. We focused on small heat shock proteins (sHSPs/HSPBs), which interact with proteins and lipids and are upregulated during aging, a major risk factor for protein misfolding diseases. HSPBs act on different microscopic steps to prevent α-synuclein aggregation, with HSPB6 showing a lipid-dependent chaperone activity. Our findings provide an example of how HSPBs diversified their mechanisms of action to reach an efficient regulation of protein misfolding and aggregation within the complex cellular environment.
    Keywords:  Biological sciences; Cell biology; Chemistry
    DOI:  https://doi.org/10.1016/j.isci.2024.110657
  20. EMBO J. 2024 Sep 16.
    Non-autophagic Golgi-LC3 lipidation facilitates TFE3 stress response against Golgi dysfunction.
    Jaemin Kang, Cathena Meiling Li, Namhoon Kim, Jongyeon Baek, Yong-Keun Jung.
      Lipidated ATG8/LC3 proteins are recruited to single membrane compartments as well as autophagosomes, supporting their functions. Although recent studies have shown that Golgi-LC3 lipidation follows Golgi damage, its molecular mechanism and function under Golgi stress remain unknown. Here, by combining DLK1 overexpression as a new strategy for induction of Golgi-specific LC3 lipidation, and the application of Golgi-damaging reagents, we unravel the mechanism and role of Golgi-LC3 lipidation. Upon DLK1 overexpression, LC3 is lipidated on the Golgi apparatus in an ATG12-ATG5-ATG16L1 complex-dependent manner; a post-Golgi trafficking blockade is the primary cause of this lipidation. During Golgi stress, ATG16L1 is recruited through its interaction with V-ATPase for Golgi-LC3 lipidation. After post-Golgi trafficking inhibition, TFE3, a key regulator of the Golgi stress response, is translocated to the nucleus. Defects in LC3 lipidation disrupt this translocation, leading to an attenuation of the Golgi stress response. Together, our results reveal the mechanism and unexplored function of Golgi-LC3 lipidation in the Golgi stress response.
    Keywords:  CASM; Golgi Apparatus; Golgi Stress Response; Post-Golgi Trafficking; V-ATPase
    DOI:  https://doi.org/10.1038/s44318-024-00233-y
  21. Elife. 2024 Sep 17. pii: RP92173. [Epub ahead of print]12
    DNL343 is an investigational CNS penetrant eukaryotic initiation factor 2B activator that prevents and reverses the effects of neurodegeneration caused by the integrated stress response.
    Ernie Yulyaningsih, Jung H Suh, Melania Fanok, Roni Chau, Hilda Solanoy, Ryan Takahashi, Anna I Bakardjiev, Isabel Becerra, N Butch Benitez, Chi-Lu Chiu, Sonnet S Davis, William E Dowdle, Timothy Earr, Anthony A Estrada, Audrey Gill, Connie Ha, Patrick C G Haddick, Kirk R Henne, Martin Larhammar, Amy W-S Leung, Romeo Maciuca, Bahram Memarzadeh, Hoang N Nguyen, Alicia A Nugent, Maksim Osipov, Yingqing Ran, Kevin Rebadulla, Elysia Roche, Thomas Sandman, Jing Wang, Joseph W Lewcock, Kimberly Scearce-Levie, Lesley A Kane, Pascal E Sanchez.
      The integrated stress response (ISR) is a conserved pathway in eukaryotic cells that is activated in response to multiple sources of cellular stress. Although acute activation of this pathway restores cellular homeostasis, intense or prolonged ISR activation perturbs cell function and may contribute to neurodegeneration. DNL343 is an investigational CNS-penetrant small-molecule ISR inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. DNL343 reduced CNS ISR activity and neurodegeneration in a dose-dependent manner in two established in vivo models - the optic nerve crush injury and an eIF2B loss of function (LOF) mutant - demonstrating neuroprotection in both and preventing motor dysfunction in the LOF mutant mouse. Treatment with DNL343 at a late stage of disease in the LOF model reversed elevation in plasma biomarkers of neuroinflammation and neurodegeneration and prevented premature mortality. Several proteins and metabolites that are dysregulated in the LOF mouse brains were normalized by DNL343 treatment, and this response is detectable in human biofluids. Several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD), a neurodegenerative disease that is driven by eIF2B LOF and chronic ISR activation, supporting their potential translational relevance. This study demonstrates that DNL343 is a brain-penetrant ISR inhibitor capable of attenuating neurodegeneration in mouse models and identifies several biomarker candidates that may be used to assess treatment responses in the clinic.
    Keywords:  biomarkers; human; integrated stress response; mouse; neurodegeneration; neuroscience
    DOI:  https://doi.org/10.7554/eLife.92173
  22. Nat Microbiol. 2024 Sep 18.
    Protein interactions in human pathogens revealed through deep learning.
    Ian R Humphreys, Jing Zhang, Minkyung Baek, Yaxi Wang, Aditya Krishnakumar, Jimin Pei, Ivan Anishchenko, Catherine A Tower, Blake A Jackson, Thulasi Warrier, Deborah T Hung, S Brook Peterson, Joseph D Mougous, Qian Cong, David Baker.
      Identification of bacterial protein-protein interactions and predicting the structures of these complexes could aid in the understanding of pathogenicity mechanisms and developing treatments for infectious diseases. Here we developed RoseTTAFold2-Lite, a rapid deep learning model that leverages residue-residue coevolution and protein structure prediction to systematically identify and structurally characterize protein-protein interactions at the proteome-wide scale. Using this pipeline, we searched through 78 million pairs of proteins across 19 human bacterial pathogens and identified 1,923 confidently predicted complexes involving essential genes and 256 involving virulence factors. Many of these complexes were not previously known; we experimentally tested 12 such predictions, and half of them were validated. The predicted interactions span core metabolic and virulence pathways ranging from post-transcriptional modification to acid neutralization to outer-membrane machinery and should contribute to our understanding of the biology of these important pathogens and the design of drugs to combat them.
    DOI:  https://doi.org/10.1038/s41564-024-01791-x
  23. J Clin Invest. 2024 Sep 17. pii: e169666. [Epub ahead of print]134(18):
    Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts.
    Hao Yang, Xiyuan Ping, Jiayue Zhou, Hailaiti Ailifeire, Jing Wu, Francisco M Nadal-Nicolás, Kiyoharu J Miyagishima, Jing Bao, Yuxin Huang, Yilei Cui, Xin Xing, Shiqiang Wang, Ke Yao, Wei Li, Xingchao Shentu.
      Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this "cataract-reversing" phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress-related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.
    Keywords:  Ophthalmology; Stem cells; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI169666
  24. Nat Commun. 2024 Sep 16. 15(1): 8119
    N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation.
    Jeremy Monroe, Daniel E Eyler, Lili Mitchell, Indrajit Deb, Abigail Bojanowski, Pooja Srinivas, Christine M Dunham, Bijoyita Roy, Aaron T Frank, Kristin S Koutmou.
      The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid and accurate protein production. Chemical modification of mRNA nucleobases can adjust the strength and pattern of this hydrogen bonding to alter protein synthesis. We investigate how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the speed and fidelity of translation. We find that m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, we also find that m1Ψ can subtly modulate the fidelity of amino acid incorporation in a codon-position and tRNA dependent manner in vitro and in human cells. Our computational modeling shows that altered energetics of mRNA:tRNA interactions largely account for the context dependence of the low levels of miscoding we observe on Ψ and m1Ψ containing codons. The outcome of translation on modified mRNA bases is thus governed by the sequence context in which they occur.
    DOI:  https://doi.org/10.1038/s41467-024-51301-0
  25. bioRxiv. 2024 Sep 07. pii: 2024.09.07.611787. [Epub ahead of print]
    Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation.
    Francesca Chandler, Poli Adi Narayana Reddy, Smita Bhutda, Rebecca L Ross, Miriam Walden, Kieran Walker, Stefano Di Donato, Joel A Cassel, Michael A Prakesch, Ahmed Aman, Alessandro Datti, Lisa J Campbell, Martina Foglizzo, Lillie Bell, Daniel N Stein, James R Ault, Rima S Al-Awar, Antonio N Calabrese, Frank Sicheri, Francesco Del Galdo, Joseph M Salvino, Roger A Greenberg, Elton Zeqiraj.
      Deubiquitylases (DUBs) play a pivotal role in cell signalling and are often regulated by homo- or hetero-interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by selectively cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). BRCC36 is a Zn2+-dependent JAMM/MPN DUB, a challenging ubiquitin protease class for the design of selective inhibitors. We identified first-in-class DUB inhibitors that act as BRISC molecular glues (BLUEs). BLUEs inhibit DUB activity by stabilising a BRISC dimer consisting of 16 subunits. The BLUE-stabilised BRISC dimer is an autoinhibited conformation, whereby the active sites and interactions with the recruiting subunit SHMT2 are blocked. This unique mode of action leads to highly selective inhibitors for BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. Structure-guided inhibitor resistant mutants confirm BLUEs on-target activity in cells, and BLUE treatment results in reduced interferon-stimulated gene (ISG) expression in human peripheral blood mononuclear cells from Scleroderma patients, a disease linked with aberrant IFNAR1 activation. BLUEs represent a new class of molecules with potential utility in Type I interferon-mediated diseases and a template for designing selective inhibitors of large protein complexes by promoting protein-protein interactions instead of blocking them.
    DOI:  https://doi.org/10.1101/2024.09.07.611787
  26. Cell Rep. 2024 Sep 14. pii: S2211-1247(24)01089-1. [Epub ahead of print]43(9): 114738
    Protein UFMylation regulates early events during ribosomal DNA-damage response.
    Pudchalaluck Panichnantakul, Lisbeth C Aguilar, Evan Daynard, Mackenzie Guest, Colten Peters, Jackie Vogel, Marlene Oeffinger.
      The highly repetitive and transcriptionally active ribosomal DNA (rDNA) genes are exceedingly susceptible to genotoxic stress. Induction of DNA double-strand breaks (DSBs) in rDNA repeats is associated with ataxia-telangiectasia-mutated (ATM)-dependent rDNA silencing and nucleolar reorganization where rDNA is segregated into nucleolar caps. However, the regulatory events underlying this response remain elusive. Here, we identify protein UFMylation as essential for rDNA-damage response in human cells. We further show the only ubiquitin-fold modifier 1 (UFM1)-E3 ligase UFL1 and its binding partner DDRGK1 localize to nucleolar caps upon rDNA damage and that UFL1 loss impairs ATM activation and rDNA transcriptional silencing, leading to reduced rDNA segregation. Moreover, analysis of nuclear and nucleolar UFMylation targets in response to DSB induction further identifies key DNA-repair factors including ATM, in addition to chromatin and actin network regulators. Taken together, our data provide evidence of an essential role for UFMylation in orchestrating rDNA DSB repair.
    Keywords:  ATRX; CP: Molecular biology; H3.3 deposition; HIRA; HUSH complex; TCOF1; UFMylation; nucleolar segregation; rDNA damage
    DOI:  https://doi.org/10.1016/j.celrep.2024.114738
  27. bioRxiv. 2024 Sep 05. pii: 2024.09.05.611450. [Epub ahead of print]
    The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation.
    Luz Saavedra-Sanchez, Mary S Dickinson, Shruti Apte, Yifeng Zhang, Maarten de Jong, Samantha Skavicus, Nicholas S Heaton, Neal M Alto, Jörn Coers.
      A central signal that marshals host defense against many infections is the lymphocyte-derived cytokine interferon-gamma (IFNγ). The IFNγ receptor is expressed on most human cells and its activation leads to the expression of antimicrobial proteins that execute diverse cell-autonomous immune programs. One such immune program consists of the sequential detection, ubiquitylation, and destruction of intracellular pathogens. Recently, the IFNγ-inducible ubiquitin E3 ligase RNF213 was identified as a pivotal mediator of such a defense axis. RNF213 provides host protection against viral, bacterial, and protozoan pathogens. To establish infections, potentially susceptible intracellular pathogens must have evolved mechanisms that subdue RNF213-controlled cell-autonomous immunity. In support of this hypothesis, we demonstrate here that a causative agent of bacillary dysentery, Shigella flexneri, uses the type III secretion system (T3SS) effector IpaH1.4 to induce the degradation of RNF213. S. flexneri mutants lacking IpaH1.4 expression are bound and ubiquitylated by RNF213 in the cytosol of IFNγ-primed host cells. Linear (M1-) and lysine-linked ubiquitin is conjugated to bacteria by RNF213 independent of the linear ubiquitin chain assembly complex (LUBAC). We find that ubiquitylation of S. flexneri is insufficient to kill intracellular bacteria, suggesting that S. flexneri employs additional virulence factors to escape from host defenses that operate downstream from RNF213-driven ubiquitylation. In brief, this study identified the bacterial IpaH1.4 protein as a direct inhibitor of mammalian RNF213 and highlights evasion of RNF213-driven immunity as a characteristic of the human-tropic pathogen Shigella.
    DOI:  https://doi.org/10.1101/2024.09.05.611450
  28. J Biol Chem. 2024 Sep 12. pii: S0021-9258(24)02276-2. [Epub ahead of print] 107775
    TBK1 adaptor AZI2/NAP1 regulates NDP52-driven mitochondrial autophagy.
    Ryu Endo, Hiroki Kinefuchi, Momoha Sawada, Reika Kikuchi, Waka Kojima, Noriyuki Matsuda, Koji Yamano.
      Damaged mitochondria are selectively eliminated in a process called mitophagy. PINK1 and Parkin amplify ubiquitin signals on damaged mitochondria, which are then recognized by autophagy adaptors to induce local autophagosome formation. NDP52 and OPTN, two essential mitophagy adaptors, facilitate de novo synthesis of pre-autophagosomal membranes near damaged mitochondria by linking ubiquitinated mitochondria and ATG8 family proteins and by recruiting core autophagy initiation components. The multifunctional serine/threonine kinase TBK1 also plays important roles in mitophagy. OPTN directly binds TBK1 to form a positive feedback loop for isolation membrane expansion. TBK1 is also thought to indirectly interact with NDP52; however, its role in NDP52-driven mitophagy remains largely unknown. Here, we focused on two TBK1 adaptors, AZI2/NAP1 and TBKBP1/SINTBAD, that are thought to mediate the TBK1-NDP52 interaction. We found that both AZI2 and TBKBP1 are recruited to damaged mitochondria during Parkin-mediated mitophagy. Further, a series of AZI2 and TBKBP1 knockout constructs combined with an OPTN knockout showed that AZI2, but not TBKBP1, impacts NDP52-driven mitophagy. In addition, we found that AZI2 at S318 is phosphorylated during mitophagy, the impairment of which slightly inhibits mitochondrial degradation. These results suggest that AZI2, in concert with TBK1, plays an important role in NDP52-driven mitophagy.
    Keywords:  autophagy; mitochondria; mitophagy; polyubiquitin chain; serine/threonine protein kinase
    DOI:  https://doi.org/10.1016/j.jbc.2024.107775
  29. Cell Rep. 2024 Sep 15. pii: S2211-1247(24)01085-4. [Epub ahead of print]43(9): 114734
    Multi-omics analysis using antibody-based in situ biotinylation technique suggests the mechanism of Cajal body formation.
    Keisuke Noguchi, Hidefumi Suzuki, Ryota Abe, Keiko Horiuchi, Rena Onoguchi-Mizutani, Nobuyoshi Akimitsu, Shintaro Ogawa, Tomohiko Akiyama, Yoko Ike, Yoko Ino, Yayoi Kimura, Akihide Ryo, Hiroshi Doi, Fumiaki Tanaka, Yutaka Suzuki, Atsushi Toyoda, Yuki Yamaguchi, Hidehisa Takahashi.
      Membrane-less subcellular compartments play important roles in various cellular functions. Although techniques exist to identify components of cellular bodies, a comprehensive method for analyzing both static and dynamic states has not been established. Here, we apply an antibody-based in situ biotinylation proximity-labeling technique to identify components of static and dynamic nuclear bodies. Using this approach, we comprehensively identify DNA, RNA, and protein components of Cajal bodies (CBs) and then clarify their interactome. By inhibiting transcription, we capture dynamic changes in CBs. Our analysis reveals that nascent small nuclear RNAs (snRNAs) transcribed in CBs contribute to CB formation by assembling RNA-binding proteins, including frontotemporal dementia-related proteins, RNA-binding motif proteins, and heterogeneous nuclear ribonucleoproteins.
    Keywords:  CP: Molecular biology; Cajal body; LLPS; biotinylation; nucleolus; small nuclear RNA; γH2AX
    DOI:  https://doi.org/10.1016/j.celrep.2024.114734
  30. Nat Commun. 2024 Sep 18. 15(1): 8184
    Structural basis of human 20S proteasome biogenesis.
    Hanxiao Zhang, Chenyu Zhou, Zarith Mohammad, Jianhua Zhao.
      New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. Here, to understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observe an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.
    DOI:  https://doi.org/10.1038/s41467-024-52513-0
  31. Commun Biol. 2024 Sep 14. 7(1): 1143
    Live-cell visualization of tau aggregation in human neurons.
    Bryan Hurtle, Christopher J Donnelly, Xin Zhang, Amantha Thathiah.
      Alzheimer's disease (AD) and more than twenty other dementias, termed tauopathies, are pathologically defined by insoluble aggregates of the microtubule-associated protein tau (MAPT). Although tau aggregation correlates with AD symptomology, the specific tau species, i.e., monomers, soluble oligomers, and insoluble aggregates that induce neurotoxicity are incompletely understood. We developed a light-responsive tau protein (optoTAU) and used viscosity-sensitive AggFluor probes to investigate the consequence(s) of tau aggregation in human neurons and identify modifiers of tau aggregation in AD and other tauopathies. We determined that optoTAU reproduces biological and structural properties of tau aggregation observed in human brains and the pathophysiological transition in tau solubility in live cells. We also provide proof-of-concept for the utilization of optoTAU as a pharmacological platform to identify modifiers of tau aggregation. These findings have broad implications for the characterization of aggregation-prone proteins and investigation of the complex relationship between protein solubility, cellular function, and disease progression.
    DOI:  https://doi.org/10.1038/s42003-024-06840-z
  32. bioRxiv. 2024 Sep 08. pii: 2024.09.04.611277. [Epub ahead of print]
    Proximity labeling defines the phagosome lumen proteome of murine and primary human macrophages.
    Benjamin L Allsup, Supriya Gharpure, Bryan D Bryson.
      Proteomic analyses of the phagosome has significantly improved our understanding of the proteins which contribute to critical phagosome functions such as apoptotic cell clearance and microbial killing. However, previous methods of isolating phagosomes for proteomic analysis have relied on cell fractionation with some intrinsic limitations. Here, we present an alternative and modular proximity-labeling based strategy for mass spectrometry proteomic analysis of the phagosome lumen, termed PhagoID. We optimize proximity labeling in the phagosome and apply PhagoID to immortalized murine macrophages as well as primary human macrophages. Analysis of proteins detected by PhagoID in murine macrophages demonstrate that PhagoID corroborates previous proteomic studies, but also nominates novel proteins with unexpected residence at the phagosome for further study. A direct comparison between the proteins detected by PhagoID between mouse and human macrophages further reveals that human macrophage phagosomes have an increased abundance of proteins involved in the oxidative burst and antigen presentation. Our study develops and benchmarks a new approach to measure the protein composition of the phagosome and validates a subset of these findings, ultimately using PhagoID to grant further insight into the core constituent proteins and species differences at the phagosome lumen.
    DOI:  https://doi.org/10.1101/2024.09.04.611277
  33. J Cell Biol. 2024 Dec 02. pii: e202401082. [Epub ahead of print]223(12):
    Feedback control of the heat shock response by spatiotemporal regulation of Hsp70.
    Rania Garde, Annisa Dea, Madeline F Herwig, Asif Ali, David Pincus.
      Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted the induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing the expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity.
    DOI:  https://doi.org/10.1083/jcb.202401082
  34. Nature. 2024 Sep 18.
    Targeted protein relocalization via protein transport coupling.
    Christine S C Ng, Aofei Liu, Bianxiao Cui, Steven M Banik.
      Subcellular protein localization regulates protein function and can be corrupted in cancers1 and neurodegenerative diseases2,3. The rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules that harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could enforce targeted protein relocalization and rewire the interactome. Here we identify a collection of shuttle proteins with potent ligands amenable to incorporation into targeted relocalization-activating molecules (TRAMs), and use these to relocalize endogenous proteins. Using a custom imaging analysis pipeline, we show that protein steady-state localization can be modulated through molecular coupling to shuttle proteins containing sufficiently strong localization sequences and expressed in the necessary abundance. We analyse the TRAM-induced relocalization of different proteins and then use nuclear hormone receptors as shuttles to redistribute disease-driving mutant proteins such as SMARCB1Q318X, TDP43ΔNLS and FUSR495X. TRAM-mediated relocalization of FUSR495X to the nucleus from the cytoplasm correlated with a reduction in the number of stress granules in a model of cellular stress. With methionyl aminopeptidase 2 and poly(ADP-ribose) polymerase 1 as endogenous cytoplasmic and nuclear shuttles, respectively, we demonstrate relocalization of endogenous PRMT9, SOS1 and FKBP12. Small-molecule-mediated redistribution of nicotinamide nucleotide adenylyltransferase 1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the genetic WldS gain-of-function phenotype in mice resistant to certain types of neurodegeneration4. The concept of targeted protein relocalization could therefore inspire approaches for treating disease through interactome rewiring.
    DOI:  https://doi.org/10.1038/s41586-024-07950-8
  35. Cancer Lett. 2024 Sep 12. pii: S0304-3835(24)00653-0. [Epub ahead of print]604 217258
    Macroautophagy/autophagy promotes resistance to KRASG12D-targeted therapy through glutathione synthesis.
    Leng Han, Lingjun Meng, Jiao Liu, Yangchun Xie, Rui Kang, Daniel J Klionsky, Daolin Tang, Yuanyuan Jia, Enyong Dai.
      KRASG12D mutation-driven pancreatic ductal adenocarcinoma (PDAC) represents a major challenge in medicine due to late diagnosis and treatment resistance. Here, we report that macroautophagy (hereafter autophagy), a cellular degradation and recycling process, contributes to acquired resistance against novel KRASG12D-targeted therapy. The KRASG12D protein inhibitor MRTX1133 induces autophagy in KRASG12D-mutated PDAC cells by blocking MTOR activity, and increased autophagic flux prevents apoptosis. Mechanistically, autophagy facilitates the generation of glutamic acid, cysteine, and glycine for glutathione synthesis. Increased glutathione levels reduce reactive oxygen species production, which impedes CYCS translocation from mitochondria to the cytosol, ultimately preventing the formation of the APAF1 apoptosome. Consequently, genetic interventions (utilizing ATG5 or BECN1 knockout) or pharmacological inhibition of autophagy (with chloroquine, bafilomycin A1, or spautin-1) enhance the anticancer activity of MRTX1133 in vitro and in various animal models (subcutaneous, patient-derived xenograft, and orthotopic). Moreover, the release of histones by apoptotic cells triggers an adaptive immune response when combining an autophagy inhibitor with MRTX1133 in immunocompetent mice. These findings establish a new strategy to overcome KRASG12D-targeted therapy resistance by inhibiting autophagy-dependent glutathione synthesis.
    Keywords:  Autophagy; Drug resistance; Glutathione; KRAS mutation; Pancreatic cancer
    DOI:  https://doi.org/10.1016/j.canlet.2024.217258
  36. bioRxiv. 2024 Sep 04. pii: 2024.09.04.611288. [Epub ahead of print]
    Genome-Wide Mapping of RNA-Protein Associations via Sequencing.
    Zhijie Qi, Shuanghong Xue, Junchen Chen, Wenxin Zhao, Kara Johnson, Xingzhao Wen, John Lalith Charles Richard, Sheng Zhong.
      RNA-protein interactions are crucial for regulating gene expression and cellular functions, with their dysregulation potentially impacting disease progression. Systematically mapping these interactions is resource-intensive due to the vast number of potential RNA and protein interactions. Here, we introduce PRIM-seq ( P rotein- R NA Interaction M apping by sequencing), a method for the concurrent de novo identification of RNA-binding proteins (RBPs) and the elucidation of their associated RNAs. PRIM-seq works by converting each RNA-protein pair into a unique chimeric DNA sequence, which is then decoded through DNA sequencing. Applied to two human cell types, PRIM-seq generated a comprehensive human RNA-protein association network (HuRPA), consisting of more than 350,000 RNA-proteins pairs involving approximately 7,000 RNAs and 11,000 proteins. The data revealed an enrichment of previously reported RBPs and RNA-protein interactions within HuRPA. We also identified LINC00339 as a protein-associating non-coding RNA and PHGDH as an RNA-associating protein. Notably, PHGDH interacts with BECN1 and ATF4 mRNAs, suppressing their protein expression and consequently inhibiting autophagy, apoptosis, and neurite outgrowth while promoting cell proliferation. PRIM-seq offers a powerful tool for discovering RBPs and RNA-protein associations, contributing to more comprehensive functional genome annotations.
    DOI:  https://doi.org/10.1101/2024.09.04.611288
  37. J Cell Biol. 2024 Dec 02. pii: e202308137. [Epub ahead of print]223(12):
    ER-plasma membrane contact sites deliver ER lipids and proteins for rapid cell surface expansion.
    Madison Smith, Lincoln Gay, Markus Babst.
      As a consequence of hypoosmotic shock, yeast cells swell rapidly and increase the surface area by ∼20% in 20 s. Approximately, 35% of this surface increase is mediated by the ER-plasma membrane contact sites, specifically the tricalbins, which are required for the delivery of both lipids and the GPI-anchored protein Crh2 from the cortical ER to the plasma membrane. Therefore, we propose a new function for the tricalbins: mediating the fusion of the ER to the plasma membrane at contact sites. This proposed fusion is triggered by calcium influx via the stretch-gated channel Cch1 and is supported by the anoctamin Ist2.
    DOI:  https://doi.org/10.1083/jcb.202308137
  38. Science. 2024 Sep 20. 385(6715): eado1868
    Large library docking identifies positive allosteric modulators of the calcium-sensing receptor.
    Fangyu Liu, Cheng-Guo Wu, Chia-Ling Tu, Isabella Glenn, Justin Meyerowitz, Anat Levit Kaplan, Jiankun Lyu, Zhiqiang Cheng, Olga O Tarkhanova, Yurii S Moroz, John J Irwin, Wenhan Chang, Brian K Shoichet, Georgios Skiniotis.
      Positive allosteric modulator (PAM) drugs enhance the activation of the calcium-sensing receptor (CaSR) and suppress parathyroid hormone (PTH) secretion. Unfortunately, these hyperparathyroidism-treating drugs can induce hypocalcemia and arrhythmias. Seeking improved modulators, we docked libraries of 2.7 million and 1.2 billion molecules against the CaSR structure. The billion-molecule docking found PAMs with a 2.7-fold higher hit rate than the million-molecule library, with hits up to 37-fold more potent. Structure-based optimization led to nanomolar leads. In ex vivo organ assays, one of these PAMs was 100-fold more potent than the standard of care, cinacalcet, and reduced serum PTH levels in mice without the hypocalcemia typical of CaSR drugs. As determined from cryo-electron microscopy structures, the PAMs identified here promote CaSR conformations that more closely resemble the activated state than those induced by the established drugs.
    DOI:  https://doi.org/10.1126/science.ado1868
  39. Neurol Genet. 2024 Oct;10(5): e200191
    Elevated VCP ATPase Activity Correlates With Disease Onset in Multisystem Proteinopathy-1.
    Sarah E Robinson, Andrew R Findlay, Shan Li, Feng Wang, Marianela Schiava, Jil Daw, Jordi Diaz-Manera, Tsui-Fen Chou, Conrad C Weihl.
       Objectives: Multisystem proteinopathy-1 (MSP1) is a late onset disease with >50 pathogenic variants in p97/VCP. MSP1 patients have multiple phenotypes that include inclusion body myopathy, Paget disease of the bone, amyotrophic lateral sclerosis, and frontotemporal dementia. There have been no clear genotype-phenotype correlations. We sought to identify genotype-phenotype correlations and associate these with VCP intrinsic ATPase activity.
    Methods: Patients with MSP1 were identified from the literature and the Cure VCP patient registry. Age at onset and at loss of ambulation were collated. VCP intrinsic ATPase activity was evaluated from recombinant purified protein.
    Results: Among the 5 most common pathogenic VCP variants in MSP1 patients, R155C patients had the earliest average age at onset (38.15 ± 9.78). This correlated with higher ATPase activity. Evaluation of 5 variants confirmed an inverse correlation between age at onset and ATPase activity (r = -0.94, p = 0.01).
    Discussion: Previous studies have reported that VCP pathogenic variants are "hyperactive." Whether this elevation in VCP ATPase activity is relevant to disease is unclear. Our study supports that in vitro VCP activity correlates with disease onset and may guide the prognosis of patients with rare or unreported variants. Moreover, it suggests that inhibition of VCP ATPase activity in MSP1 may be therapeutic.
    DOI:  https://doi.org/10.1212/NXG.0000000000200191
  40. Nat Chem Biol. 2024 Sep 19.
    A tRNA modification with aminovaleramide facilitates AUA decoding in protein synthesis.
    Kenjyo Miyauchi, Satoshi Kimura, Naho Akiyama, Kazuki Inoue, Kensuke Ishiguro, Thien-Son Vu, Veerasak Srisuknimit, Kenta Koyama, Gosuke Hayashi, Akiko Soma, Asuteka Nagao, Mikako Shirouzu, Akimitsu Okamoto, Matthew K Waldor, Tsutomu Suzuki.
      Modified tRNA anticodons are critical for proper mRNA translation during protein synthesis. It is generally thought that almost all bacterial tRNAsIle use a modified cytidine-lysidine (L)-at the first position (34) of the anticodon to decipher the AUA codon as isoleucine (Ile). Here we report that tRNAsIle from plant organelles and a subset of bacteria contain a new cytidine derivative, designated 2-aminovaleramididine (ava2C). Like L34, ava2C34 governs both Ile-charging ability and AUA decoding. Cryo-electron microscopy structural analyses revealed molecular details of codon recognition by ava2C34 with a specific interaction between its terminal amide group and an mRNA residue 3'-adjacent to the AUA codon. These findings reveal the evolutionary variation of an essential tRNA modification and demonstrate the molecular basis of AUA decoding mediated by a unique tRNA modification.
    DOI:  https://doi.org/10.1038/s41589-024-01726-x
  41. Cell Rep. 2024 Sep 18. pii: S2211-1247(24)01098-2. [Epub ahead of print]43(10): 114747
    Regulation of synapse density by Pumilio RNA-binding proteins.
    Lisa K Randolph, Michaela M Pauers, José C Martínez, Leslie J Sibener, Michael A Zrzavy, Nyle A Sharif, Tatiana M Gonzalez, Kapil V Ramachandran, Daniel Dominguez, Ulrich Hengst.
      The formation, stabilization, and elimination of synapses are tightly regulated during neural development and into adulthood. Pumilio RNA-binding proteins regulate the translation and localization of many synaptic mRNAs and are developmentally downregulated in the brain. We found that simultaneous downregulation of Pumilio 1 and 2 increases both excitatory and inhibitory synapse density in primary hippocampal neurons and promotes synapse maturation. Loss of Pum1 and Pum2 in the mouse brain was associated with an increase in mRNAs involved in mitochondrial function and synaptic translation. These findings reveal a role for developmental Pumilio downregulation as a permissive step in the maturation of synapses and suggest that modulation of Pumilio levels is a cell-intrinsic mechanism by which neurons tune their capacity for synapse stabilization.
    Keywords:  CP: Molecular biology; CP: Neuroscience; Pumilio; RNA-binding proteins; local translation; mRNA regulation; synapse
    DOI:  https://doi.org/10.1016/j.celrep.2024.114747
  42. Mol Biol Cell. 2024 Sep 18. mbcE24050226
    Spatial and signaling overlap of growth factor receptor systems at clathrin-coated sites.
    Marco A Alfonzo-Méndez, Marie-Paule Strub, Justin W Taraska.
      Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, and endocytic proteins. Yet, the composition and control of these complexes in response to external cues remain unclear. We use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures in human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with growth factors. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR1, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR activity with drugs prevents the recruitment of both EGFR and FGFR1. EGF was able to activate FGFR1 phosphorylation. Our data reveals novel co-clustering and activation of receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF or FGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.
    DOI:  https://doi.org/10.1091/mbc.E24-05-0226
  43. Nat Cell Biol. 2024 Sep 17.
    Plasma membrane curvature regulates the formation of contacts with the endoplasmic reticulum.
    Yang Yang, Luis A Valencia, Chih-Hao Lu, Melissa L Nakamoto, Ching-Ting Tsai, Chun Liu, Huaxiao Yang, Wei Zhang, Zeinab Jahed, Wan-Ru Lee, Francesca Santoro, Jen Liou, Joseph C Wu, Bianxiao Cui.
      Contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM) play a crucial role in governing calcium regulation and lipid homeostasis. Despite their significance, the factors regulating their spatial distribution on the PM remain elusive. Inspired by observations in cardiomyocytes, where ER-PM contact sites concentrate on tubular PM invaginations known as transverse tubules, we hypothesize that PM curvature plays a role in ER-PM contact formation. Through precise control of PM invaginations, we show that PM curvatures locally induce the formation of ER-PM contacts in cardiomyocytes. Intriguingly, the junctophilin family of ER-PM tethering proteins, specifically expressed in excitable cells, is the key player in this process, whereas the ubiquitously expressed extended synaptotagmin-2 does not show a preference for PM curvature. At the mechanistic level, we find that the low-complexity region (LCR) and membrane occupation and recognition nexus (MORN) motifs of junctophilins can bind independently to the PM, but both the LCR and MORN motifs are required for targeting PM curvatures. By examining the junctophilin interactome, we identify a family of curvature-sensing proteins-Eps15 homology domain-containing proteins-that interact with the MORN_LCR motifs and facilitate the preferential tethering of junctophilins to curved PM. These findings highlight the pivotal role of PM curvature in the formation of ER-PM contacts in cardiomyocytes and unveil a mechanism for the spatial regulation of ER-PM contacts through PM curvature modulation.
    DOI:  https://doi.org/10.1038/s41556-024-01511-x
  44. STAR Protoc. 2024 Sep 18. pii: S2666-1667(24)00486-6. [Epub ahead of print]5(4): 103321
    Protocol for detecting glycoRNAs using metabolic labeling and northwestern blot.
    Luoyi Li, Ningning Zhang, Carla Pantoja, Yadong Wang, Jun Lu.
      GlycoRNAs are glycosylated RNAs that can be detected in many cell types and often partly reside on the outer cell surface, with a recently demonstrated role in mediating neutrophil-endothelium interaction. Here, we present a protocol for glycoRNA detection based on metabolic tracing and northwestern blot. We describe steps for metabolic labeling of cells, extraction and purification of RNA, biotin labeling of RNA, and northwestern blot for glycoRNA detection. We also incorporate optimized conditions for biotin labeling, RNA dye, and membrane blocking. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.
    Keywords:  Cell Biology; Cell culture; Chemistry; Molecular Biology; Molecular/Chemical Probes
    DOI:  https://doi.org/10.1016/j.xpro.2024.103321
  45. J Cell Biol. 2024 Nov 04. pii: e202312119. [Epub ahead of print]223(11):
    Migfilin promotes autophagic flux through direct interaction with SNAP29 and Vamp8.
    Renwei Cai, Panzhu Bai, Meiling Quan, Yanyan Ding, Wenjie Wei, Chengmin Liu, Aihua Yang, Zailin Xiong, Guizhen Li, Binbin Li, Yi Deng, Ruijun Tian, Yan G Zhao, Chuanyue Wu, Ying Sun.
      Autophagy plays a crucial role in cancer cell survival by facilitating the elimination of detrimental cellular components and the recycling of nutrients. Understanding the molecular regulation of autophagy is critical for developing interventional approaches for cancer therapy. In this study, we report that migfilin, a focal adhesion protein, plays a novel role in promoting autophagy by increasing autophagosome-lysosome fusion. We found that migfilin is associated with SNAP29 and Vamp8, thereby facilitating Stx17-SNAP29-Vamp8 SNARE complex assembly. Depletion of migfilin disrupted the formation of the SNAP29-mediated SNARE complex, which consequently blocked the autophagosome-lysosome fusion, ultimately suppressing cancer cell growth. Restoration of the SNARE complex formation rescued migfilin-deficiency-induced autophagic flux defects. Finally, we found depletion of migfilin inhibited cancer cell proliferation. SNARE complex reassembly successfully reversed migfilin-deficiency-induced inhibition of cancer cell growth. Taken together, our study uncovers a new function of migfilin as an autophagy-regulatory protein and suggests that targeting the migfilin-SNARE assembly could provide a promising therapeutic approach to alleviate cancer progression.
    DOI:  https://doi.org/10.1083/jcb.202312119
  46. Elife. 2024 Sep 16. pii: RP96643. [Epub ahead of print]13
    PPI-hotspotID for detecting protein-protein interaction hot spots from the free protein structure.
    Yao Chi Chen, Karen Sargsyan, Jon D Wright, Yu-Hsien Chen, Yi-Shuian Huang, Carmay Lim.
      Experimental detection of residues critical for protein-protein interactions (PPI) is a time-consuming, costly, and labor-intensive process. Hence, high-throughput PPI-hot spot prediction methods have been developed, but they have been validated using relatively small datasets, which may compromise their predictive reliability. Here, we introduce PPI-hotspotID, a novel method for identifying PPI-hot spots using the free protein structure, and validated it on the largest collection of experimentally confirmed PPI-hot spots to date. We explored the possibility of detecting PPI-hot spots using (i) FTMap in the PPI mode, which identifies hot spots on protein-protein interfaces from the free protein structure, and (ii) the interface residues predicted by AlphaFold-Multimer. PPI-hotspotID yielded better performance than FTMap and SPOTONE, a webserver for predicting PPI-hot spots given the protein sequence. When combined with the AlphaFold-Multimer-predicted interface residues, PPI-hotspotID yielded better performance than either method alone. Furthermore, we experimentally verified several PPI-hotspotID-predicted PPI-hot spots of eukaryotic elongation factor 2. Notably, PPI-hotspotID can reveal PPI-hot spots not obvious from complex structures, including those in indirect contact with binding partners. PPI-hotspotID serves as a valuable tool for understanding PPI mechanisms and aiding drug design. It is available as a web server (https://ppihotspotid.limlab.dnsalias.org/) and open-source code (https://github.com/wrigjz/ppihotspotid/).
    Keywords:  AlphaFold-Multimer; AutoGluon; PPI-hot spots; free protein structure; machine learning; molecular biophysics; none; protein binding; structural biology
    DOI:  https://doi.org/10.7554/eLife.96643
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