bims-proteo 2024-06-02 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–06–02
forty papers selected by
Eric Chevet, INSERM

CUL-6/cullin ubiquitin ligase-mediated degradation of HSP-90 by intestinal lysosomes promotes thermotolerance.
RTN3L and CALCOCO1 function in parallel to maintain proteostasis in the endoplasmic reticulum.
TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.
IGHMBP2 deletion suppresses translation and activates the integrated stress response.
Tissue-specific landscape of protein aggregation and quality control in an aging vertebrate.
Proteome birthdating reveals age-selectivity of protein ubiquitination.
YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.
Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions.
K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors.
Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy.
Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.
Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.
Movement of the endoplasmic reticulum is driven by multiple classes of vesicles marked by Rab-GTPases.
FIP200 phosphorylation regulates late steps in mitophagy.
Mitochondrial dysfunction heightens the integrated stress response to drive ALS pathogenesis.
Structure-based discovery of CFTR potentiators and inhibitors.
Islet cell stress induced by insulin-degrading enzyme deficiency promotes regeneration and protection from autoimmune diabetes.
Mass spectral feature analysis of ubiquitylated peptides provides insights into probing the dark ubiquitylome.
Structural insights into GrpEL1-mediated nucleotide and substrate release of human mitochondrial Hsp70.
IKZF2 Degradation: It's Time to Take into Account it When Designing Cereblon-based PROTACs.
CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy.
Small molecule induced STING degradation facilitated by the HECT ligase HERC4.
Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.
Stress granule-localized USP8 potentiates cGAS-mediated type I interferonopathies through deubiquitination of DDX3X.
Proximal protein landscapes of the type I interferon signaling cascade reveal negative regulation by PJA2.
Identification of protein aggregates in the aging vertebrate brain with prion-like and phase-separation properties.
Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation.
Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.
A protein sequence-based deep transfer learning framework for identifying human proteome-wide deubiquitinase-substrate interactions.
UFL1 Regulates Cellular Homeostasis by Targeting Endoplasmic Reticulum and Mitochondria in NEFA-stimulated Bovine Mammary Epithelial Cells via the IRE1α/XBP1 pathway.
The hidden world of protein aggregation.
Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements.
Lysineless HiBiT and NanoLuc Tagging Systems as Alternative Tools Monitoring Targeted Protein Degradation.
TRP14 is the rate-limiting enzyme for intracellular cystine reduction and regulates proteome cysteinylation.
Conformations of Bcs1L undergoing ATP hydrolysis suggest a concerted translocation mechanism for folded iron-sulfur protein substrate.
Interleukin-1 mediated hyperinflammation in XIAP-deficiency is associated with defective autophagy.
De novo design of buttressed loops for sculpting protein functions.
The balance between antiviral and antibacterial responses during M. tuberculosis infection is regulated by the ubiquitin ligase CBL.
PolyQ-expanded ataxin-2 aggregation impairs cellular processing-body homeostasis via sequestering the RNA helicase DDX6.
Integrative modeling meets deep learning: Recent advances in modeling protein assemblies.

Cell Rep. 2024 May 24. pii: S2211-1247(24)00607-7. [Epub ahead of print]43(6): 114279

CUL-6/cullin ubiquitin ligase-mediated degradation of HSP-90 by intestinal lysosomes promotes thermotolerance.

Mario Bardan Sarmiento, Spencer S Gang, Patricija van Oosten-Hawle, Emily R Troemel.

  Heat shock can be a lethal stressor. Previously, we described a CUL-6/cullin-ring ubiquitin ligase complex in the nematode Caenorhabditis elegans that is induced by intracellular intestinal infection and proteotoxic stress and that promotes improved survival upon heat shock (thermotolerance). Here, we show that CUL-6 promotes thermotolerance by targeting the heat shock protein HSP-90 for degradation. We show that CUL-6-mediated lowering of HSP-90 protein levels, specifically in the intestine, improves thermotolerance. Furthermore, we show that lysosomal function is required for CUL-6-mediated promotion of thermotolerance and that CUL-6 directs HSP-90 to lysosome-related organelles upon heat shock. Altogether, these results indicate that a CUL-6 ubiquitin ligase promotes organismal survival upon heat shock by promoting HSP-90 degradation in intestinal lysosomes. Thus, HSP-90, a protein commonly associated with protection against heat shock and promoting degradation of other proteins, is itself degraded to protect against heat shock.

Keywords:  C. elegans; CP: Cell biology; CUL-6; HSF-1; HSP-90; cullin-ring ubiquitin ligase; intracellular pathogen response; lysosome; lysosome-related organelles; proteostasis; thermotolerance

DOI:  https://doi.org/10.1016/j.celrep.2024.114279
Autophagy. 2024 May 31. 1-9

RTN3L and CALCOCO1 function in parallel to maintain proteostasis in the endoplasmic reticulum.

Kamal Kumar, Ravi Chidambaram, Smriti Parashar, Susan Ferro-Novick.

  Reticulophagy is mediated by autophagy receptors that function in one of the two domains of the ER, tubules or flat sheets. Three different conserved mammalian receptors mediate autophagy in ER tubules: RTN3L, ATL3 and CALCOCO1. Previous studies have shown that RTN3L maintains proteostasis by targeting mutant aggregation-prone proteins for autophagy at distinct foci in ER tubules that we named ERPHS (ER-reticulophagy sites). The role for ATL3 and CALCOCO1 in proteostasis has not been addressed. Here we analyzed three different misfolded disease-causing RTN3L substrates and show that ATL3 and CALCOCO1 target the same cargoes for autophagy. Colocalization and knock down studies revealed that RTN3L and ATL3 are both required for the formation of RTN3L-containing ERPHS, while CALCOCO1 is not. We propose that RTN3L, ATL3 and CALCOCO1 work in parallel to maintain proteostasis within the ER network by targeting cargoes at different sites in the tubules.Abbreviation ATL3: atlastin GTPase 3; Baf: bafilomycin A1; CALCOCO1: calcium binding and coiled-coil domain 1; Epr1: ER-phagy receptor 1; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERPHS: ER-reticulophagy sites; LAMP1: lysosomal associated membrane protein 1; PGRMC1: progesterone receptor membrane component 1; POMC: proopiomelanocortin; Pro-AVP: pro-arginine vasopressin; RETREG1: reticulophagy regulator 1; reticulophagy: endoplasmic reticulum selective autophagy; RTN3L: reticulon 3 long isoform; VAPA: VAMP associated protein A.

Keywords:  Aggregation-prone proteins; endoplasmic reticulum; endoplasmic reticulum stress; reticulophagy receptors; selective autophagy

DOI:  https://doi.org/10.1080/15548627.2024.2353502
Cell Rep. 2024 May 29. pii: S2211-1247(24)00622-3. [Epub ahead of print]43(6): 114294

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Bhaskar Saha, Hallvard Olsvik, Geneva L Williams, Seeun Oh, Gry Evjen, Eva Sjøttem, Michael A Mandell.

  Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.

Keywords:  CP: Cell biology; NBR1; NDP52; Optineurin; TAX1BP1; TBK1; TRIM27/RFP; autophagy; p62; tripartite motif; ubiquitin ligase

DOI:  https://doi.org/10.1016/j.celrep.2024.114294
Life Sci Alliance. 2024 Aug;pii: e202302554. [Epub ahead of print]7(8):

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

Jesslyn Park, Hetvee Desai, José M Liboy-Lugo, Sohyun Gu, Ziad Jowhar, Albert Xu, Stephen N Floor.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

DOI: https://doi.org/10.26508/lsa.202302554
Dev Cell. 2024 May 24. pii: S1534-5807(24)00266-1. [Epub ahead of print]

Tissue-specific landscape of protein aggregation and quality control in an aging vertebrate.

Yiwen R Chen, Itamar Harel, Param Priya Singh, Inbal Ziv, Eitan Moses, Uri Goshtchevsky, Ben E Machado, Anne Brunet, Daniel F Jarosz.

  Protein aggregation is a hallmark of age-related neurodegeneration. Yet, aggregation during normal aging and in tissues other than the brain is poorly understood. Here, we leverage the African turquoise killifish to systematically profile protein aggregates in seven tissues of an aging vertebrate. Age-dependent aggregation is strikingly tissue specific and not simply driven by protein expression differences. Experimental interrogation in killifish and yeast, combined with machine learning, indicates that this specificity is linked to protein-autonomous biophysical features and tissue-selective alterations in protein quality control. Co-aggregation of protein quality control machinery during aging may further reduce proteostasis capacity, exacerbating aggregate burden. A segmental progeria model with accelerated aging in specific tissues exhibits selectively increased aggregation in these same tissues. Intriguingly, many age-related protein aggregates arise in wild-type proteins that, when mutated, drive human diseases. Our data chart a comprehensive landscape of protein aggregation during vertebrate aging and identify strong, tissue-specific associations with dysfunction and disease.

Keywords:  aggregates; aging; multi-tissue; protein homeostasis; proteomics; systems biology

DOI:  https://doi.org/10.1016/j.devcel.2024.04.014
Mol Cell Proteomics. 2024 May 24. pii: S1535-9476(24)00081-1. [Epub ahead of print] 100791

Proteome birthdating reveals age-selectivity of protein ubiquitination.

Michael E Meadow, Sarah Broas, Margaret Hoare, Fatemeh Alimohammadi, Kevin A Welle, Kyle Swovick, Jennifer R Hryhorenko, John C Martinez, Seyed Ali Biashad, Andrei Seluanov, Vera Gorbunova, Abigail Buchwalter, Sina Ghaemmaghami.

  Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed "proteome birthdating" that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics from a single biological sample encoding multiple labeling time-points. As a first application of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identify a smaller subset of older ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways.

Keywords:  Aging; Mass spectrometry (MS); Protein turnover; Proteomics; Proteostasis; Ubiquitin Proteasome System (UPS)

DOI:  https://doi.org/10.1016/j.mcpro.2024.100791
EMBO J. 2024 May 31.

YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.

Shinri Kitta, Tatsuya Kaminishi, Momoko Higashi, Takayuki Shima, Kohei Nishino, Nobuhiro Nakamura, Hidetaka Kosako, Tamotsu Yoshimori, Akiko Kuma.

  The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.

Keywords:  Autophagy Receptor; Golgi Turnover; Golgiphagy; Organelle Degradation; Selective Autophagy

DOI:  https://doi.org/10.1038/s44318-024-00131-3
Dev Cell. 2024 May 27. pii: S1534-5807(24)00322-8. [Epub ahead of print]

Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions.

Max Koppers, Nazmiye Özkan, Ha H Nguyen, Daphne Jurriens, Janine McCaughey, Dan T M Nguyen, Chun Hei Li, Riccardo Stucchi, Maarten Altelaar, Harold D MacGillavry, Lukas C Kapitein, Casper C Hoogenraad, Ginny G Farías.

  Local mRNA translation in axons is critical for the spatiotemporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons; however, how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation in developing rat hippocampal cultured neurons. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with axonal ER tubules in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation and binds to mRNAs enriched for axonal membrane proteins. Importantly, the impairment of axonal ER-ribosome interactions causes defects in axon morphology. Our results establish a role for the axonal ER in dynamically localizing mRNA translation, which is important for proper neuron development.

Keywords:  ER shape; ER-based translation; P180/RRBP1; axonal ER; axonal protein synthesis; endoplasmic reticulum; local translation; mRNA localization; neuron development; ribosomes

DOI:  https://doi.org/10.1016/j.devcel.2024.05.005
Life Sci Alliance. 2024 Aug;pii: e202402740. [Epub ahead of print]7(8):

K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors.

Anita Waltho, Oliver Popp, Christopher Lenz, Lukas Pluska, Mahil Lambert, Volker Dötsch, Philipp Mertins, Thomas Sommer.

The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different lengths, linkage types, and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying lengths, as well as homotypic and heterotypic branched chains of the two most abundant linkage types-lysine 48-linked (K48) and lysine 63-linked (K63) Ub. We identified some of the first K48/K63-linked branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4, and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50, and p97 adaptor FAF1. Crucially, we compared datasets collected using two common deubiquitinase inhibitors-chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.

DOI: https://doi.org/10.26508/lsa.202402740
Autophagy. 2024 May 31. 1-16

Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy.

Kelong Ma, Wei Xian, Hongtao Liu, Rundong Shu, Jinli Ge, Zhao-Qing Luo, Xiaoyun Liu, Jiazhang Qiu.

  Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB.Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: Legionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin.

Keywords:  DEPTOR; deubiquitinase; effector protein; intracellular bacteria; type IV secretion system; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2024.2353492
Cell Chem Biol. 2024 May 27. pii: S2451-9456(24)00180-6. [Epub ahead of print]

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Valentina Rossio, Joao A Paulo, Xinyue Liu, Steven P Gygi, Randall W King.

  Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Keywords:  DUB substrates; TMT-proteomics; Xenopus; deubiquitylating enzymes; ubiquitin

DOI:  https://doi.org/10.1016/j.chembiol.2024.05.001
Genes Dev. 2024 May 30.

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

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

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

Keywords:  HSF-1; PPARα; heat shock response; protein aggregation

DOI:  https://doi.org/10.1101/gad.351829.124
bioRxiv. 2024 May 15. pii: 2024.05.14.592021. [Epub ahead of print]

Movement of the endoplasmic reticulum is driven by multiple classes of vesicles marked by Rab-GTPases.

Allison Langley, Sarah Abeling-Wang, Erinn Wagner, John Salogiannis.

Peripheral endoplasmic reticulum (ER) tubules move along microtubules to interact with various organelles through membrane contact sites (MCS). Traditionally, ER moves by either sliding along stable microtubules via molecular motors or attaching to the plus ends of dynamic microtubules through tip attachment complexes (TAC). A recently discovered third process, hitchhiking, involves motile vesicles pulling ER tubules along microtubules. Previous research showed that ER hitchhikes on Rab5- and Rab7-marked endosomes, but it is uncertain if other Rab-vesicles can do the same. In U2OS cells, we screened Rabs for their ability to cotransport with ER tubules and found that ER hitchhikes on post-Golgi vesicles marked by Rab6 (isoforms a and b). Rab6-ER hitchhiking occurs independently of ER-endolysosome contacts and TAC-mediated ER movement. Disrupting either Rab6 or the motility of Rab6-vesicles reduces overall ER movement. Conversely, relocating these vesicles to the cell periphery causes peripheral ER accumulation, indicating that Rab6-vesicle motility is crucial for a subset of ER movements. Proximal post-Golgi vesicles marked by TGN46 are involved in Rab6-ER hitchhiking, while other post-Golgi vesicles (Rabs 8/10/11/13/14) are not essential for ER movement. Our further analysis finds that ER to Golgi vesicles marked by Rab1 are also capable of driving a subset of ER movements. Taken together, our findings suggest that ER hitchhiking on Rab-vesicles is a significant mode of ER movement.
SIGNIFICANCE STATEMENT: Peripheral endoplasmic reticulum tubules move on microtubules by either attaching to motors (cargo adaptor-mediated), dynamic microtubule-plus ends (tip attachment complexes) or motile vesicles (hitchhiking) but the prevalence of each mode is not clearPost-Golgi vesicles marked by Rab6/TGN46 and ER to Golgi vesicles marked by Rab1 drive ER movementsER hitchhiking on multiple classes of vesicles (endolysosomal, post-Golgi and ER to Golgi) marked by Rabs plays a prominent role in ER movement.

DOI: https://doi.org/10.1101/2024.05.14.592021
J Mol Biol. 2024 May 29. pii: S0022-2836(24)00226-2. [Epub ahead of print] 168631

FIP200 phosphorylation regulates late steps in mitophagy.

Christopher Eickhorst, Riccardo Babic, Jorrell Rush-Kittle, Leon Lucya, Fatimah Lami Imam, Pablo Sánchez-Martín, David M Hollenstein, Jonas Michaelis, Christian Münch, Chris Meisinger, Dea Slade, Laura Gámez-Díaz, Claudine Kraft.

  Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.

Keywords:  ATG16L1; Atg1/ULK1 kinase complex; FIP200; autophagy; mitophagy

DOI:  https://doi.org/10.1016/j.jmb.2024.168631
bioRxiv. 2024 May 14. pii: 2024.05.13.594000. [Epub ahead of print]

Mitochondrial dysfunction heightens the integrated stress response to drive ALS pathogenesis.

Curran Landry, James Costanzo, Miguel Mitne-Neto, Mayana Zatz, Ashleigh Schaffer, Maria Hatzoglou, Alysson Muotri, Helen Cristina Miranda.

Vesicle-associated membrane protein-associated protein-B (VAPB) is an ER membrane bound protein. VAPB P56S causes a dominant, familial form of amyotrophic lateral sclerosis (ALS), however, the mechanism through which this mutation causes motor neuron (MN) disease remains unknown. Using inducible wild type (WT) and VAPB P56S expressing iPSC-derived MNs we show that VAPB P56S, but not WT, protein decreased neuronal firing and mitochondrial-ER contact (MERC) with an associated age-dependent decrease in mitochondrial membrane potential (MMP); all typical characteristics of MN-disease. We further show that VAPB P56S expressing iPSC-derived MNs have enhanced age-dependent sensitivity to ER stress. We identified elevated expression of the master regulator of the Integrated Stress Response (ISR) marker ATF4 and decreased protein synthesis in the VAPB P56S iPSC-derived MNs. Chemical inhibition of ISR with the compound, ISRIB, rescued all MN disease phenotype in VAPB P56S MNs. Thus, our results not only support ISR inhibition as a potential therapeutic target for ALS patients, but also provides evidence to pathogenesis.

DOI: https://doi.org/10.1101/2024.05.13.594000
Cell. 2024 May 23. pii: S0092-8674(24)00472-0. [Epub ahead of print]

Structure-based discovery of CFTR potentiators and inhibitors.

Fangyu Liu, Anat Levit Kaplan, Jesper Levring, Jürgen Einsiedel, Stephanie Tiedt, Katharina Distler, Natalie S Omattage, Ivan S Kondratov, Yurii S Moroz, Harlan L Pietz, John J Irwin, Peter Gmeiner, Brian K Shoichet, Jue Chen.

  The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Keywords:  ABC transporter; anion channel; inhibitors; large-scale docking; ligand discovery; potentiators

DOI:  https://doi.org/10.1016/j.cell.2024.04.046
iScience. 2024 Jun 21. 27(6): 109929

Islet cell stress induced by insulin-degrading enzyme deficiency promotes regeneration and protection from autoimmune diabetes.

Shuaishuai Zhu, Emmanuelle Waeckel-Énée, Masaya Oshima, Anna Moser, Marie-Andrée Bessard, Abdelaziz Gdoura, Kevin Roger, Nina Mode, Joanna Lipecka, Ayse Yilmaz, Barbara Bertocci, Julien Diana, Benjamin Saintpierre, Ida Chiara Guerrera, Raphael Scharfmann, Stefania Francesconi, François-Xavier Mauvais, Peter van Endert.

  Tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with variable demand for insulin. Here, we asked how insulin-degrading enzyme (IDE) affects beta cell adaptation to metabolic and immune stress. C57BL/6 and autoimmune non-obese diabetic (NOD) mice lacking IDE were exposed to proteotoxic, metabolic, and immune stress. IDE deficiency induced a low-level UPR with islet hypertrophy at the steady state, rapamycin-sensitive beta cell proliferation enhanced by proteotoxic stress, and beta cell decompensation upon high-fat feeding. IDE deficiency also enhanced the UPR triggered by proteotoxic stress in human EndoC-βH1 cells. In Ide-/- NOD mice, islet inflammation specifically induced regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. These findings establish a role of IDE in islet cell protein homeostasis, demonstrate how its absence induces metabolic decompensation despite beta cell proliferation, and UPR-independent islet regeneration in the presence of inflammation.

Keywords:  Molecular biology; Physiology; Proteomics; Transcriptomics

DOI:  https://doi.org/10.1016/j.isci.2024.109929
bioRxiv. 2024 May 17. pii: 2024.05.16.594577. [Epub ahead of print]

Mass spectral feature analysis of ubiquitylated peptides provides insights into probing the dark ubiquitylome.

Regina M Edgington, Damien Beau Wilburn.

Ubiquitylation is a structurally and functionally diverse post translational modification that involves the covalent attachment of the small protein ubiquitin to other protein substrates. Trypsin-based proteomics is the most common approach for globally identifying ubiquitylation sites. However, we estimate that such methods are unable to detect ~40% of ubiquitylation sites in the human proteome - i.e., the dark ubiquitylome - including many important for human health and disease. In this meta-analysis of three large ubiquitylomic datasets, we performed a series of bioinformatic analyses to assess experimental features that could aid in uniquely identifying site-specific ubiquitylation events. Spectral predictions from Prosit were compared to experimental spectra of tryptic ubiquitylated peptides, revealing previously uncharacterized fragmentation of the diGly scar. Analysis of the LysC-derived ubiquitylated peptides reveal systematic, multidimensional peptide fragmentation, including diagnostic b-ions from fragmentation of the LysC ubiquitin scar. Comprehensively, these findings provide diagnostic spectral signatures of modification events that could applied to new analysis methods for non-tryptic ubiquitylomics.

DOI: https://doi.org/10.1101/2024.05.16.594577
bioRxiv. 2024 May 13. pii: 2024.05.10.593630. [Epub ahead of print]

Structural insights into GrpEL1-mediated nucleotide and substrate release of human mitochondrial Hsp70.

Marc A Morizono, Kelly L McGuire, Natalie I Birouty, Mark A Herzik.

Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70 (Hsp70) system. In human mitochondria, mitochondrial Hsp70 (mortalin) and the nucleotide exchange factor (GrpEL1) work synergistically to stabilize proteins, assemble protein complexes, and facilitate protein import. However, our understanding of the molecular mechanisms guiding these processes is hampered by limited structural information. To elucidate these mechanistic details, we used cryoEM to determine the first structures of full-length human mortalin-GrpEL1 complexes in previously unobserved states. Our structures and molecular dynamics simulations allow us to delineate specific roles for mortalin-GrpEL1 interfaces and to identify steps in GrpEL1-mediated nucleotide and substrate release by mortalin. Subsequent analyses reveal conserved mechanisms across bacteria and mammals and facilitate a complete understanding of sequential nucleotide and substrate release for the Hsp70 chaperone system.

DOI: https://doi.org/10.1101/2024.05.10.593630
Chembiochem. 2024 May 27. e202400365

IKZF2 Degradation: It's Time to Take into Account it When Designing Cereblon-based PROTACs.

Minglei Li.

  Proteolysis-targeting chimera (PROTAC) has become a very important means of protein degradation and a new way of disease treatment. In particular, PROTACs constructed with ligands for E3 ligase cereblon account for more than 90% of the PROTACs currently in clinical research. Notably, CRBN ligands themselves are a class of molecular glue compounds capable of degrading neo-substrate proteins. Compared to the target proteins degradation, the degradation of neo-substrates, especially IKZF2, has not received enough attention. Therefore, this review summarizes the currently published IKZF2 degraders derived from articles and patents, which are conducive to the design of PROTACs with desired IKZF2 degradation from the perspective of medicinal chemistry.

Keywords:  IKZF2; PROTACs; cereblon; molecular glue; protein degradation

DOI:  https://doi.org/10.1002/cbic.202400365
Sci Adv. 2024 May 31. 10(22): eadn2050

CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy.

Marta Zaninello, Tim Schlegel, Hendrik Nolte, Mujeeb Pirzada, Elisa Savino, Esther Barth, Ines Klein, Hauke Wüstenberg, Tesmin Uddin, Lisa Wolff, Brunhilde Wirth, Helmar C Lehmann, Jean-Michel Cioni, Thomas Langer, Elena I Rugarli.

Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.

DOI: https://doi.org/10.1126/sciadv.adn2050
Nat Commun. 2024 May 29. 15(1): 4584

Small molecule induced STING degradation facilitated by the HECT ligase HERC4.

Merve Mutlu, Isabel Schmidt, Andrew I Morrison, Benedikt Goretzki, Felix Freuler, Damien Begue, Oliver Simic, Nicolas Pythoud, Erik Ahrne, Sandra Kapps, Susan Roest, Debora Bonenfant, Delphine Jeanpierre, Thi-Thanh-Thao Tran, Rob Maher, Shaojian An, Amandine Rietsch, Florian Nigsch, Andreas Hofmann, John Reece-Hoyes, Christian N Parker, Danilo Guerini.

Stimulator of interferon genes (STING) is a central component of the cytosolic nucleic acids sensing pathway and as such master regulator of the type I interferon response. Due to its critical role in physiology and its' involvement in a variety of diseases, STING has been a focus for drug discovery. Targeted protein degradation (TPD) has emerged as a promising pharmacology for targeting previously considered undruggable proteins by hijacking the cellular ubiquitin proteasome system (UPS) with small molecules. Here, we identify AK59 as a STING degrader leveraging HERC4, a HECT-domain E3 ligase. Additionally, our data reveals that AK59 is effective on the common pathological STING mutations, suggesting a potential clinical application of this mechanism. Thus, these findings introduce HERC4 to the fields of TPD and of compound-induced degradation of STING, suggesting potential therapeutic applications.

DOI: https://doi.org/10.1038/s41467-024-48922-w
ArXiv. 2024 May 15. pii: arXiv:2405.09699v1. [Epub ahead of print]

Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.

Prashant Kaushal, Manisha R Ummadi, Gwendolyn M Jang, Yennifer Delgado, Sara K Makanani, Sophie F Blanc, Decan M Winters, Jiewei Xu, Benjamin Polacco, Yuan Zhou, Erica Stevenson, Manon Eckhardt, Lorena Zuliani-Alvarez, Robyn Kaake, Danielle L Swaney, Nevan Krogan, Mehdi Bouhaddou.

Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity tagged bait proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity tagged bait gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas.
Cell Rep. 2024 May 24. pii: S2211-1247(24)00576-X. [Epub ahead of print]43(6): 114248

Stress granule-localized USP8 potentiates cGAS-mediated type I interferonopathies through deubiquitination of DDX3X.

Xuejing Zhang, Lulu Han, Jinxiu Hou, Huiyu Yang, Haiyan Xu, Guosheng Li, Qiang Shu, Deyu Zhu, Yi Zheng, Chengjiang Gao.

  Cyclic GMP-AMP synthase (cGAS) undergoes liquid-liquid phase separation (LLPS) to trigger downstream signaling upon double-stranded DNA (dsDNA) stimulation, and the condensed cGAS colocalizes with stress granules (SGs). However, the molecular mechanism underlying the modulation of cGAS activation by SGs remains elusive. In this study, we show that USP8 is localized to SGs upon dsDNA stimulation and potentiates cGAS-stimulator of interferon genes (STING) signaling. A USP8 inhibitor ameliorates pathological inflammation in Trex1-/- mice. Systemic lupus erythematosus (SLE) databases indicate a positive correlation between USP8 expression and SLE. Mechanistic study shows that the SG protein DDX3X promotes cGAS phase separation and activation in a manner dependent on its intrinsic LLPS. USP8 cleaves K27-linked ubiquitin chains from the intrinsically disordered region (IDR) of DDX3X to enhance its condensation. In conclusion, we demonstrate that USP8 catalyzes the deubiquitination of DDX3X to facilitate cGAS condensation and activation and that inhibiting USP8 is a promising strategy for alleviating cGAS-mediated autoimmune diseases.

Keywords:  CP: Molecular biology

DOI:  https://doi.org/10.1016/j.celrep.2024.114248
Nat Commun. 2024 May 27. 15(1): 4484

Proximal protein landscapes of the type I interferon signaling cascade reveal negative regulation by PJA2.

Samira Schiefer, Benjamin G Hale.

Deciphering the intricate dynamic events governing type I interferon (IFN) signaling is critical to unravel key regulatory mechanisms in host antiviral defense. Here, we leverage TurboID-based proximity labeling coupled with affinity purification-mass spectrometry to comprehensively map the proximal human proteomes of all seven canonical type I IFN signaling cascade members under basal and IFN-stimulated conditions. This uncovers a network of 103 high-confidence proteins in close proximity to the core members IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, and validates several known constitutive protein assemblies, while also revealing novel stimulus-dependent and -independent associations between key signaling molecules. Functional screening further identifies PJA2 as a negative regulator of IFN signaling via its E3 ubiquitin ligase activity. Mechanistically, PJA2 interacts with TYK2 and JAK1, promotes their non-degradative ubiquitination, and limits the activating phosphorylation of TYK2 thereby restraining downstream STAT signaling. Our high-resolution proximal protein landscapes provide global insights into the type I IFN signaling network, and serve as a valuable resource for future exploration of its functional complexities.

DOI: https://doi.org/10.1038/s41467-024-48800-5
Cell Rep. 2024 May 24. pii: S2211-1247(23)00798-2. [Epub ahead of print] 112787

Identification of protein aggregates in the aging vertebrate brain with prion-like and phase-separation properties.

Itamar Harel, Yiwen R Chen, Inbal Ziv, Param Priya Singh, Daniel Heinzer, Paloma Navarro Negredo, Uri Goshtchevsky, Wei Wang, Gwendoline Astre, Eitan Moses, Andrew McKay, Ben E Machado, Katja Hebestreit, Sifei Yin, Alejandro Sánchez Alvarado, Daniel F Jarosz, Anne Brunet.

  Protein aggregation, which can sometimes spread in a prion-like manner, is a hallmark of neurodegenerative diseases. However, whether prion-like aggregates form during normal brain aging remains unknown. Here, we use quantitative proteomics in the African turquoise killifish to identify protein aggregates that accumulate in old vertebrate brains. These aggregates are enriched for prion-like RNA-binding proteins, notably the ATP-dependent RNA helicase DDX5. We validate that DDX5 forms aggregate-like puncta in the brains of old killifish and mice. Interestingly, DDX5's prion-like domain allows these aggregates to propagate across many generations in yeast. In vitro, DDX5 phase separates into condensates. Mutations that abolish DDX5 prion propagation also impair the protein's ability to phase separate. DDX5 condensates exhibit enhanced enzymatic activity, but they can mature into inactive, solid aggregates. Our findings suggest that protein aggregates with prion-like properties form during normal brain aging, which could have implications for the age-dependency of cognitive decline.

Keywords:  CP: Cell biology; CP: Neuroscience; N. furzeri; aggregates; aging; killifish; phase separation

DOI:  https://doi.org/10.1016/j.celrep.2023.112787
bioRxiv. 2024 May 14. pii: 2024.05.10.593557. [Epub ahead of print]

Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation.

Tiancheng Hu, Yiping Liu, Jeremy Fleck, Cason King, Elaine Schalk, Zhenyu Zhang, Andrew Mehle, Judith A Smith.

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-β expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-β induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-β, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-β mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-β via mitochondrial dsDNA release.

DOI: https://doi.org/10.1101/2024.05.10.593557
bioRxiv. 2024 May 14. pii: 2024.05.14.593897. [Epub ahead of print]

Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.

Megan Jr Yeo, Olivia Zhang, Xiaowen Xie, Eunju Nam, Neil Connor Payne, Pallavi M Gosavi, Hui Si Kwok, Irtiza Iram, Ceejay Lee, Jiaming Li, Nicholas J Chen, Hanjie Jiang, Zhipeng A Wang, Kwangwoon Lee, Haibin Mao, Stefan A Harry, Idris A Barakat, Mariko Takahashi, Amanda L Waterbury, Marco Barone, Andrea Mattevi, Liron Bar-Peled, Philip A Cole, Ralph Mazitschek, Brian B Liau, Ning Zheng.

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

DOI: https://doi.org/10.1101/2024.05.14.593897
Nat Commun. 2024 May 28. 15(1): 4519

A protein sequence-based deep transfer learning framework for identifying human proteome-wide deubiquitinase-substrate interactions.

Yuan Liu, Dianke Li, Xin Zhang, Simin Xia, Yingjie Qu, Xinping Ling, Yang Li, Xiangren Kong, Lingqiang Zhang, Chun-Ping Cui, Dong Li.

Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by "wet lab" experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.

DOI: https://doi.org/10.1038/s41467-024-48446-3
Free Radic Biol Med. 2024 May 29. pii: S0891-5849(24)00483-0. [Epub ahead of print]

UFL1 Regulates Cellular Homeostasis by Targeting Endoplasmic Reticulum and Mitochondria in NEFA-stimulated Bovine Mammary Epithelial Cells via the IRE1α/XBP1 pathway.

YeXiao Yan, JunPeng Huang, XiangXing Chen, Yuan Li, WeiGuo Zhao, ChengMin Li.

  Elevated levels of NEFA caused by negative energy balance in transition cows induce cellular dyshomeostasis. Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) can maintain cellular homeostasis and act as a critical regulator of stress responses besides functioning in the ubiquitin-like system. The objective of this study was to elucidate the UFL1 working mechanism on promoting cellular adaptations in bovine mammary epithelial cells (BMECs) in response to NEFA challenge, with an emphasis on the ER and mitochondrial function. The results showed that exogenous NEFA and UFL1 depletion resulted in the disorder of ER and mitochondrial homeostasis and the damage of BMEC integrity, overexpression of UFL1 effectively alleviated the NEFA-induced cellular dyshomeostasis. Mechanistically, our study found that UFL1 had a strong interaction with IRE1α and could modulate the IRE1α/XBP1 pathway of unfolded protein response in NEFA-stimulated BMECs, thereby contributing to the modulation of cellular homeostasis. These findings imply that targeting UFL1 may be a therapeutic alternative to relieve NEB-induced metabolic changes in perinatal dairy cows.

Keywords:  IRE1α/XBP1 pathway; NEFA; UFL1; bovine mammary epithelial cells; cellular homeostasis

DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.05.039
Prog Mol Biol Transl Sci. 2024 ;pii: S1877-1173(24)00069-3. [Epub ahead of print]206 473-494

The hidden world of protein aggregation.

Bahareh Dabirmanesh, Khosro Khajeh, Vladimir N Uversky.

  Though the book's journey into The Hidden World of Protein Aggregation has come to an end, the search for knowledge, the development of healthier lives, and the discovery of nature's mysteries continue, promising new horizons and discoveries yet to be discovered. The intricacies of protein misfolding and aggregation remain a mystery in cellular biology, despite advances made in unraveling them. In this chapter, we will summarize the specific conclusions from the previous chapters and explore the persistent obstacles and unanswered questions that motivate scientists to pursue exploration of protein misfolding and aggregation.

Keywords:  Biotechnological approach; Functional amyloids; Protein aggregation; Therapeutic approach

DOI:  https://doi.org/10.1016/bs.pmbts.2024.03.014
Nat Commun. 2024 May 27. 15(1): 4504

Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements.

Ravi Dhiman, Rehani S Perera, Chetan S Poojari, Haakon T A Wiedemann, Reinhard Kappl, Christopher W M Kay, Jochen S Hub, Bianca Schrul.

Lipid droplet (LD) function relies on proteins partitioning between the endoplasmic reticulum (ER) phospholipid bilayer and the LD monolayer membrane to control cellular adaptation to metabolic changes. It has been proposed that these hairpin proteins integrate into both membranes in a similar monotopic topology, enabling their passive lateral diffusion during LD emergence at the ER. Here, we combine biochemical solvent-accessibility assays, electron paramagnetic resonance spectroscopy and intra-molecular crosslinking experiments with molecular dynamics simulations, and determine distinct intramembrane positionings of the ER/LD protein UBXD8 in ER bilayer and LD monolayer membranes. UBXD8 is deeply inserted into the ER bilayer with a V-shaped topology and adopts an open-shallow conformation in the LD monolayer. Major structural rearrangements are required to enable ER-to-LD partitioning. Free energy calculations suggest that such structural transition is unlikely spontaneous, indicating that ER-to-LD protein partitioning relies on more complex mechanisms than anticipated and providing regulatory means for this trans-organelle protein trafficking.

DOI: https://doi.org/10.1038/s41467-024-48843-8
bioRxiv. 2024 May 17. pii: 2024.05.14.594249. [Epub ahead of print]

Lysineless HiBiT and NanoLuc Tagging Systems as Alternative Tools Monitoring Targeted Protein Degradation.

Hanfeng Lin, Kristin Riching, May Poh Lai, Dong Lu, Ran Cheng, Xiaoli Qi, Jin Wang.

Target protein degradation (TPD) has emerged as a revolutionary approach in drug discovery, leveraging the cell's intrinsic machinery to selectively degrade disease-associated proteins. Proteolysis-Targeting Chimeras (PROTACs) exemplify this strategy, exploiting heterobifunctional molecules to induce ubiquitination and subsequent degradation of target proteins. The clinical advancement of PROTACs underscores their potential in therapeutic intervention, with numerous projects progressing through clinical stages. However, monitoring subtle changes in protein abundance induced by TPD molecules demands highly sensitive assays. Nano-luciferase (nLuc) fusion proteins, or the NanoBiT technology derived from it, offer a robust screening platform due to their high sensitivity and stability. Despite these advantages, concerns have arisen regarding potential degradation artifacts introduced by tagging systems due to the presence of lysine residues on them, prompting the development of alternative tools. In this study, we introduce HiBiT-RR and nLuc K0 , variants devoid of lysine residues, to mitigate such artifacts. Our findings demonstrate that HiBiT-RR maintains similar sensitivity and binding affinity with the original HiBiT. Moreover, the comparison between nLuc WT and nLuc K0 constructs reveals variations in degradation patterns induced by certain PROTAC molecules, emphasizing the importance of choosing appropriate tagging systems to ensure the reliability of experimental outcomes in studying protein degradation processes.

DOI: https://doi.org/10.1101/2024.05.14.594249
EMBO J. 2024 May 29.

TRP14 is the rate-limiting enzyme for intracellular cystine reduction and regulates proteome cysteinylation.

Pablo Martí-Andrés, Isabela Finamor, Isabel Torres-Cuevas, Salvador Pérez, Sergio Rius-Pérez, Hildegard Colino-Lage, David Guerrero-Gómez, Esperanza Morato, Anabel Marina, Patrycja Michalska, Rafael León, Qing Cheng, Eszter Petra Jurányi, Klaudia Borbényi-Galambos, Iván Millán, Péter Nagy, Antonio Miranda-Vizuete, Edward E Schmidt, Antonio Martínez-Ruiz, Elias Sj Arnér, Juan Sastre.

  It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans.

Keywords:  Acute Pancreatitis; Cysteine Homeostasis; Protein Cysteinylation; Proteotoxic Stress; Transsulfuration

DOI:  https://doi.org/10.1038/s44318-024-00117-1
Nat Commun. 2024 May 31. 15(1): 4655

Conformations of Bcs1L undergoing ATP hydrolysis suggest a concerted translocation mechanism for folded iron-sulfur protein substrate.

Jingyu Zhan, Allison Zeher, Rick Huang, Wai Kwan Tang, Lisa M Jenkins, Di Xia.

The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form.

DOI: https://doi.org/10.1038/s41467-024-49029-y
Blood. 2024 May 31. pii: blood.2023023707. [Epub ahead of print]

Interleukin-1 mediated hyperinflammation in XIAP-deficiency is associated with defective autophagy.

Dilan Dissanayake, Ashkan Firouzabady, Mohammad Massumi, Guillermo A de Paz Linares, Christian Marshall, Spencer A Freeman, Ronald Laxer, Rae S M Yeung.

Deficiency of X-linked Inhibitor of Apoptosis Protein (XIAP) is a rare genetic condition that can present with recurrent episodes of hemophagocytic lymphohistiocytosis (HLH), though the exact mechanisms leading to this hyperinflammatory disorder are unclear. Understanding its biology is critical to developing targeted therapies for this potentially fatal disease. Here we report on a novel multi-exonic intragenic duplication leading to XIAP deficiency with recurrent HLH that demonstrated complete response to interleukin (IL)-1b blockade. We further demonstrate using both primary patient cells and genetically modified THP-1 monocyte cell lines that, contrary to what has previously been shown in mouse cells, XIAP-deficient human macrophages do not produce excess IL-1b when stimulated under standard conditions. Instead, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated hyperproduction of IL-1b is observed only when the XIAP-deficient cells are stimulated under autophagy-promoting conditions and this correlates with defective autophagic flux as measured by decreased accumulation of the early autophagy marker LC3-II. This work therefore highlights IL-1b blockade as a therapeutic option for patients with XIAP deficiency experiencing recurrent HLH and identifies a critical role for XIAP in promoting autophagy as a means of limiting IL-1b-mediated hyperinflammation during periods of cellular stress.

DOI: https://doi.org/10.1182/blood.2023023707
Nat Chem Biol. 2024 May 30.

De novo design of buttressed loops for sculpting protein functions.

Hanlun Jiang, Kevin M Jude, Kejia Wu, Jorge Fallas, George Ueda, T J Brunette, Derrick R Hicks, Harley Pyles, Aerin Yang, Lauren Carter, Mila Lamb, Xinting Li, Paul M Levine, Lance Stewart, K Christopher Garcia, David Baker.

In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9-14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.

DOI: https://doi.org/10.1038/s41589-024-01632-2
bioRxiv. 2024 May 23. pii: 2024.05.15.594178. [Epub ahead of print]

The balance between antiviral and antibacterial responses during M. tuberculosis infection is regulated by the ubiquitin ligase CBL.

Tina Truong, Kelsey E Martin, Michelle R Salemi, Abigail E Ray, Brett S Phinney, Bennett H Penn.

As a first line of host defense, macrophages must be able to effectively sense and respond to diverse types of pathogens, and while a particular type of immune response may be beneficial in some circumstances, it can be detrimental in others. Upon infecting a macrophage, M. tuberculosis ( Mtb ) induces proinflammatory cytokines that activate antibacterial responses. Surprisingly, Mtb also triggers antiviral responses that actually hinder the ability of macrophages to control Mtb infection. The ubiquitin ligase CBL suppresses these antiviral responses and shifts macrophages toward a more antibacterial state during Mtb infection, however, the mechanisms by which CBL regulates immune signaling are unknown. We found that CBL controls responses to multiple stimuli and broadly suppresses the expression of antiviral effector genes. We then used mass-spectrometry to investigate potential CBL substrates and identified over 46,000 ubiquitylated peptides in Mtb -infected macrophages, as well as roughly 400 peptides with CBL35 dependent ubiquitylation. We then performed genetic interaction analysis of CBL and its putative substrates, and identified the Fas associated factor 2 (FAF2) adapter protein as a key signaling molecule protein downstream of CBL. Together, these analyses identify thousands of new ubiquitin-mediated signaling events during the innate immune response and reveal an important new regulatory hub in this response.

DOI: https://doi.org/10.1101/2024.05.15.594178
J Biol Chem. 2024 May 27. pii: S0021-9258(24)01914-8. [Epub ahead of print] 107413

PolyQ-expanded ataxin-2 aggregation impairs cellular processing-body homeostasis via sequestering the RNA helicase DDX6.

Jian-Yang Wang, Ya-Jun Liu, Xiang-Le Zhang, Yin-Hu Liu, Lei-Lei Jiang, Hong-Yu Hu.

  Ataxin-2 (Atx2) is a polyglutamine (polyQ) tract-containing RNA-binding protein, while its polyQ expansion may cause protein aggregation that is implicated in the pathogenesis of neurodegenerative diseases such as spinocerebellar ataxia type 2 (SCA2). However, the molecular mechanism underlying how Atx2 aggregation contributes to the proteinopathies remains elusive. Here, we investigated the influence of Atx2 aggregation on the assembly and functionality of cellular processing bodies (P-bodies) by using biochemical and fluorescence imaging approaches. We have revealed that polyQ-expanded (PQE) Atx2 sequesters the DEAD-box RNA helicase (DDX6), an essential component of P-bodies, into aggregates or puncta via some RNA sequences. The N-terminal like-Sm (LSm) domain of Atx2 (residues 82-184) and the C-terminal helicase domain of DDX6 are responsible for the interaction and specific sequestration. Moreover, sequestration of DDX6 may aggravate pre-mRNA mis-splicing, and interfere with the assembly of cellular P-bodies, releasing the endoribonuclease MARF1 that promotes mRNA decay and translational repression. Rescuing the DDX6 protein level can recover the assembly and functionality of P-bodies preventing targeted mRNA from degradation. This study provides a line of evidence for sequestration of the P-body components and impairment of the P-body homeostasis in dysregulating RNA metabolism, which is implicated in the disease pathologies and a potential therapeutic target.

Keywords:  Aggregation; Ataxin-2; DDX6; P-bodies; Sequestration; Translational repression; mRNA decay

DOI:  https://doi.org/10.1016/j.jbc.2024.107413
Curr Opin Struct Biol. 2024 May 24. pii: S0959-440X(24)00068-X. [Epub ahead of print]87 102841

Integrative modeling meets deep learning: Recent advances in modeling protein assemblies.

Ben Shor, Dina Schneidman-Duhovny.

Recent progress in protein structure prediction based on deep learning revolutionized the field of Structural Biology. Beyond single proteins, it also enabled high-throughput prediction of structures of protein-protein interactions. Despite the success in predicting complex structures, large macromolecular assemblies still require specialized approaches. Here we describe recent advances in modeling macromolecular assemblies using integrative and hierarchical approaches. We highlight applications that predict protein-protein interactions and challenges in modeling complexes based on the interaction networks, including the prediction of complex stoichiometry and heterogeneity.

DOI: https://doi.org/10.1016/j.sbi.2024.102841