bims-proteo Biomed News
on Proteostasis
Issue of 2023‒03‒12
thirty-six papers selected by
Eric Chevet
INSERM
  1. COP I and II dependent trafficking controls ER-associated degradation in mammalian cells.
  2. Chemoproteomics-enabled discovery of a covalent molecular glue degrader targeting NF-κB.
  3. Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin.
  4. Regional and age-dependent changes in ubiquitination in cellular and mouse models of Spinocerebellar ataxia type 3.
  5. TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.
  6. A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease.
  7. Activation of Gcn2 by small molecules designed to be inhibitors.
  8. Regulation of germline proteostasis by HSF1 and insulin/IGF-1 signaling.
  9. The E3 ubiquitin ligases regulate inflammation in cardiovascular diseases.
  10. A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.
  11. Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.
  12. STING directly recruits WIPI2 for autophagosome formation during STING-induced autophagy.
  13. BAG6 supports stress fiber formation by preventing the ubiquitin-mediated degradation of RhoA.
  14. Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.
  15. Phosphorylation-linked complex profiling identifies assemblies required for Hippo signal integration.
  16. The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA.
  17. Differential role for rapid proteostasis in Rho GTPase-mediated control of quiescent endothelial integrity.
  18. Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria.
  19. PP6 regulation of Aurora A-TPX2 limits NDC80 phosphorylation and mitotic spindle size.
  20. The tumor suppressor functions of ubiquitin ligase KPC1: from cell cycle control to NF-κB regulator.
  21. Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target.
  22. Inhibition of nonsense-mediated decay induces nociceptive sensitization through activation of the integrated stress response.
  23. An insect viral protein disrupts stress granule formation in mammalian cells.
  24. Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.
  25. CDK13 phosphorylates the translation machinery and promotes tumorigenic protein synthesis.
  26. Novel approaches to targeted protein degradation technologies in drug discovery.
  27. Coupling substrate-trapping with proximity-labeling to identify protein tyrosine phosphatase PTP1B signaling networks.
  28. ARE-PROTACs Enable Co-degradation of an Nrf2-MafG Heterodimer.
  29. Identifying multiscale translational safety biomarkers using a network-based systems approach.
  30. Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.
  31. Translation re-initiation after uORFs does not fully protect mRNAs from nonsense-mediated decay.
  32. Sustained overexpression of spliced X-box-binding protein-1 in neurons leads to spontaneous seizures and sudden death in mice.
  33. Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains.
  34. De novo design of small beta barrel proteins.
  35. Stressing out over mucus secretion.
  36. RNA-binding protein FXR1 drives cMYC translation by recruiting eIF4F complex to the translation start site.
  1. iScience. 2023 Mar 17. 26(3): 106232
    COP I and II dependent trafficking controls ER-associated degradation in mammalian cells.
    Navit Ogen-Shtern, Chieh Chang, Haddas Saad, Niv Mazkereth, Chaitanya Patel, Marina Shenkman, Gerardo Z Lederkremer.
      Misfolded proteins and components of the endoplasmic reticulum (ER) quality control and ER associated degradation (ERAD) machineries concentrate in mammalian cells in the pericentriolar ER-derived quality control compartment (ERQC), suggesting it as a staging ground for ERAD. By tracking the chaperone calreticulin and an ERAD substrate, we have now determined that the trafficking to the ERQC is reversible and recycling back to the ER is slower than the movement in the ER periphery. The dynamics suggest vesicular trafficking rather than diffusion. Indeed, using dominant negative mutants of ARF1 and Sar1 or the drugs Brefeldin A and H89, we observed that COPI inhibition causes accumulation in the ERQC and increases ERAD, whereas COPII inhibition has the opposite effect. Our results suggest that targeting of misfolded proteins to ERAD involves COPII-dependent transport to the ERQC and that they can be retrieved to the peripheral ER in a COPI-dependent manner.
    Keywords:  Cell; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2023.106232
  2. Cell Chem Biol. 2023 Mar 04. pii: S2451-9456(23)00057-0. [Epub ahead of print]
    Chemoproteomics-enabled discovery of a covalent molecular glue degrader targeting NF-κB.
    Elizabeth A King, Yoojin Cho, Nathan S Hsu, Dustin Dovala, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Daniel K Nomura.
      Targeted protein degradation has arisen as a powerful therapeutic modality for degrading disease targets. While proteolysis-targeting chimera (PROTAC) design is more modular, the discovery of molecular glue degraders has been more challenging. Here, we have coupled the phenotypic screening of a covalent ligand library with chemoproteomic approaches to rapidly discover a covalent molecular glue degrader and associated mechanisms. We have identified a cysteine-reactive covalent ligand EN450 that impairs leukemia cell viability in a NEDDylation and proteasome-dependent manner. Chemoproteomic profiling revealed covalent interaction of EN450 with an allosteric C111 in the E2 ubiquitin-conjugating enzyme UBE2D. Quantitative proteomic profiling revealed the degradation of the oncogenic transcription factor NFKB1 as a putative degradation target. Our study thus puts forth the discovery of a covalent molecular glue degrader that uniquely induced the proximity of an E2 with a transcription factor to induce its degradation in cancer cells.
    Keywords:  E2 ligase; NFKB1; UBE2D; activity-based protein profiling; molecular glue; targeted protein degradation; transcription factor
    DOI:  https://doi.org/10.1016/j.chembiol.2023.02.008
  3. iScience. 2023 Mar 17. 26(3): 106150
    Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin.
    Susan J Qualls-Histed, Casey P Nielsen, Jason A MacGurn.
      Glucose transporters are gatekeepers of cellular glucose metabolism. Understanding how their activity is regulated can provide insight into mechanisms of glucose homeostasis and diseases arising from dysregulation of glucose transport. Glucose stimulates endocytosis of the human glucose transporter GLUT1, but several important questions remain surrounding the intracellular trafficking itinerary of GLUT1. Here, we report that increased glucose availability triggers lysosomal trafficking of GLUT1 in HeLa cells, with a subpopulation of GLUT1 routed through ESCRT-associated late endosomes. This itinerary requires the arrestin-like protein TXNIP, which interacts with both clathrin and E3 ubiquitin ligases to promote GLUT1 lysosomal trafficking. We also find that glucose stimulates GLUT1 ubiquitylation, which promotes its lysosomal trafficking. Our results suggest that excess glucose first triggers TXNIP-mediated endocytosis of GLUT1 and, subsequently, ubiquitylation to promote lysosomal trafficking. Our findings underscore how complex coordination of multiple regulators is required for fine-tuning of GLUT1 stability at the cell surface.
    Keywords:  Biological sciences; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2023.106150
  4. bioRxiv. 2023 Feb 02. pii: 2023.02.01.526671. [Epub ahead of print]
    Regional and age-dependent changes in ubiquitination in cellular and mouse models of Spinocerebellar ataxia type 3.
    Haiyang Luo, Sokol V Todi, Henry L Paulson, Maria do Carmo Costa.
      Spinocerebellar ataxia type 3 (SCA3), also known as Machadoâ€"Joseph disease, is the most common dominantly inherited ataxia. SCA3 is caused by a CAG repeat expansion in the ATXN3 gene that encodes an expanded tract of polyglutamine (polyQ) in the disease protein ataxin-3 (ATXN3). As a deubiquitinating enzyme, ATXN3 regulates numerous cellular processes including proteasome- and autophagy-mediated protein degradation. In SCA3 disease brain, polyQ-expanded ATXN3 accumulates with other cellular constituents, including ubiquitin (Ub)-modified proteins, in select areas like the cerebellum and the brainstem, but whether pathogenic ATXN3 affects the abundance of ubiquitinated species is unknown. Here, in mouse and cellular models of SCA3, we investigated whether elimination of murine Atxn3 or expression of wild-type or polyQ-expanded human ATXN3 alters soluble levels of overall ubiquitination, as well as K48-linked (K48-Ub) and K63-linked (K63-Ub) chains. Levels of ubiquitination were assessed in the cerebellum and brainstem of 7- and 47-week-old Atxn3 knockout and SCA3 transgenic mice, and also in relevant mouse and human cell lines. In older mice, we observed that wild-type ATXN3 impacts the cerebellar levels of K48-Ub proteins. In contrast, pathogenic ATXN3 leads to decreased brainstem abundance of K48-Ub species in younger mice and changes in both cerebellar and brainstem K63-Ub levels in an age-dependent manner: younger SCA3 mice have higher levels of K63-Ub while older mice have lower levels of K63-Ub compared to controls. Human SCA3 neuronal progenitor cells also show a relative increase in K63-Ub proteins upon autophagy inhibition. We conclude that wild-type and mutant ATXN3 differentially impact K48-Ub- and K63-Ub-modified proteins in the brain in a region- and age-dependent manner.
    DOI:  https://doi.org/10.1101/2023.02.01.526671
  5. Mol Cell. 2023 Mar 02. pii: S1097-2765(23)00150-8. [Epub ahead of print]
    TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.
    François Le Guerroué, Eric N Bunker, William M Rosencrans, Jack T Nguyen, Mohammed A Basar, Achim Werner, Tsui-Fen Chou, Chunxin Wang, Richard J Youle.
      Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.
    Keywords:  FIP200; FIR; LIR; Selective autophagy; TAX1BP1; TBK1; TNIP1; mitophagy; mitophagy regulation
    DOI:  https://doi.org/10.1016/j.molcel.2023.02.023
  6. EMBO J. 2023 Mar 10. e113033
    A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease.
    Yu Cao, Jing Zheng, Huayun Wan, Yuqiu Sun, Song Fu, Shanshan Liu, Baiyu He, Gaihong Cai, Yang Cao, Huanwei Huang, Qi Li, Yan Ma, She Chen, Fengchao Wang, Hui Jiang.
      Mitophagy is a fundamental quality control mechanism of mitochondria. Its regulatory mechanisms and pathological implications remain poorly understood. Here, via a mitochondria-targeted genetic screen, we found that knockout (KO) of FBXL4, a mitochondrial disease gene, hyperactivates mitophagy at basal conditions. Subsequent counter screen revealed that FBXL4-KO hyperactivates mitophagy via two mitophagy receptors BNIP3 and NIX. We determined that FBXL4 functions as an integral outer-membrane protein that forms an SCF-FBXL4 ubiquitin E3 ligase complex. SCF-FBXL4 ubiquitinates BNIP3 and NIX to target them for degradation. Pathogenic FBXL4 mutations disrupt SCF-FBXL4 assembly and impair substrate degradation. Fbxl4-/- mice exhibit elevated BNIP3 and NIX proteins, hyperactive mitophagy, and perinatal lethality. Importantly, knockout of either Bnip3 or Nix rescues metabolic derangements and viability of the Fbxl4-/- mice. Together, beyond identifying SCF-FBXL4 as a novel mitochondrial ubiquitin E3 ligase restraining basal mitophagy, our results reveal hyperactivated mitophagy as a cause of mitochondrial disease and suggest therapeutic strategies.
    Keywords:  BNIP3/NIX; FBXL4; mitochondrial disease; mitophagy; ubiquitin-proteasome pathway
    DOI:  https://doi.org/10.15252/embj.2022113033
  7. J Biol Chem. 2023 Mar 08. pii: S0021-9258(23)00237-5. [Epub ahead of print] 104595
    Activation of Gcn2 by small molecules designed to be inhibitors.
    KennethR Carlson, MillieM Georgiadis, Feven Tameire, KirkA Staschke, RonaldC Wek.
      The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.
    Keywords:  Gcn2; Integrated stress response; eIF2 kinase; eIF2 phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2023.104595
  8. Biochem Soc Trans. 2023 Mar 09. pii: BST20220616. [Epub ahead of print]
    Regulation of germline proteostasis by HSF1 and insulin/IGF-1 signaling.
    Tahir Muhammad, Jian Li.
      Protein homeostasis (proteostasis) is essential for cellular function and organismal health and requires the concerted actions of protein synthesis, folding, transport, and turnover. In sexually reproducing organisms, the immortal germline lineage passes genetic information across generations. Accumulating evidence indicates the importance of proteome integrity for germ cells as genome stability. As gametogenesis involves very active protein synthesis and is highly energy-demanding, it has unique requirements for proteostasis regulation and is sensitive to stress and nutrient availability. The heat shock factor 1 (HSF1), a key transcriptional regulator of cellular response to cytosolic and nuclear protein misfolding has evolutionarily conserved roles in germline development. Similarly, insulin/insulin-like growth factor-1 (IGF-1) signaling, a major nutrient-sensing pathway, impacts many aspects of gametogenesis. Here, we focus on HSF1 and IIS to review insights into their roles in germline proteostasis and discuss the implications on gamete quality control during stress and aging.
    Keywords:  HSF1; gametogenesis; insulin/IGF-1 signaling; proteostasis; stress response
    DOI:  https://doi.org/10.1042/BST20220616
  9. Semin Cell Dev Biol. 2023 Mar 03. pii: S1084-9521(23)00035-6. [Epub ahead of print]
    The E3 ubiquitin ligases regulate inflammation in cardiovascular diseases.
    Xiao Chen, Jia Ma, Zhi-Wei Wang, Zhiting Wang.
      Accumulating evidence has illustrated that the E3 ubiquitin ligases critically participate in the development and progression of cardiovascular diseases. Dysregulation of E3 ubiquitin ligases exacerbates cardiovascular diseases. Blockade or activation of E3 ubiquitin ligases mitigates cardiovascular performance. Therefore, in this review, we mainly introduced the critical role and underlying molecular mechanisms of E3 ubiquitin ligase NEDD4 family in governing the initiation and progression of cardiovascular diseases, including ITCH, WWP1, WWP2, Smurf1, Smurf2, Nedd4-1 and Nedd4-2. Moreover, the functions and molecular insights of other E3 ubiquitin ligases, such as F-box proteins, in cardiovascular disease development and malignant progression are described. Furthermore, we illustrate several compounds that alter the expression of E3 ubiquitin ligases to alleviate cardiovascular diseases. Therefore, modulation of E3 ubiquitin ligases could be a novel and promising strategy for improvement of therapeutic efficacy of deteriorative cardiovascular diseases.
    Keywords:  Cardiovascular disease; Degradation; E3 ligases, Inflammation; Heart
    DOI:  https://doi.org/10.1016/j.semcdb.2023.02.008
  10. Dev Cell. 2023 Mar 06. pii: S1534-5807(23)00049-7. [Epub ahead of print]
    A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.
    Elaiyaraja Subramanian, Ahmed Elewa, Gonçalo Brito, Anoop Kumar, Åsa Segerstolpe, Christos Karampelias, Åsa Björklund, Rickard Sandberg, Karen Echeverri, Weng-Onn Lui, Olov Andersson, András Simon.
      Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.
    Keywords:  blastema; cell state; miRNA; muscle; newt; stem cells
    DOI:  https://doi.org/10.1016/j.devcel.2023.02.007
  11. J Extracell Vesicles. 2023 Mar;12(3): e12311
    Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.
    Pauline P Marie, Shih-Jung Fan, John Mason, Adam Wells, Cláudia C Mendes, S Mark Wainwright, Sheherezade Scott, Roman Fischer, Adrian L Harris, Clive Wilson, Deborah C I Goberdhan.
      Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in late Rab7-positive multivesicular endosomes, and also in recycling Rab11a-positive endosomes, particularly under some forms of nutrient stress. The core proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) participate in exosome biogenesis and ILV-mediated destruction of ubiquitinylated cargos. Accessory ESCRT-III components have reported roles in ESCRT-III-mediated vesicle scission, but their precise functions are poorly defined. They frequently only appear essential under stress. Comparative proteomics analysis of human small extracellular vesicles revealed that accessory ESCRT-III proteins, CHMP1A, CHMP1B, CHMP5 and IST1, are increased in Rab11a-enriched exosome preparations. We show that these proteins are required to form ILVs in Drosophila secondary cell recycling endosomes, but unlike core ESCRTs, they are not involved in degradation of ubiquitinylated proteins in late endosomes. Furthermore, CHMP5 knockdown in human HCT116 colorectal cancer cells selectively inhibits Rab11a-exosome production. Accessory ESCRT-III knockdown suppresses seminal fluid-mediated reproductive signalling by secondary cells and the growth-promoting activity of Rab11a-exosome-containing EVs from HCT116 cells. We conclude that accessory ESCRT-III components have a specific, ubiquitin-independent role in Rab11a-exosome generation, a mechanism that might be targeted to selectively block pro-tumorigenic activities of these vesicles in cancer.
    Keywords:  CHMP5; ESCRT; Rab11a-exosome; extracellular vesicle; recycling endosome
    DOI:  https://doi.org/10.1002/jev2.12311
  12. EMBO J. 2023 Mar 06. e112387
    STING directly recruits WIPI2 for autophagosome formation during STING-induced autophagy.
    Wei Wan, Chuying Qian, Qian Wang, Jin Li, Hongtao Zhang, Lei Wang, Maomao Pu, Yewei Huang, Zhengfu He, Tianhua Zhou, Han-Ming Shen, Wei Liu.
      The cGAS-STING pathway plays an important role in host defense by sensing pathogen DNA, inducing type I IFNs, and initiating autophagy. However, the molecular mechanism of autophagosome formation in cGAS-STING pathway-induced autophagy is still unclear. Here, we report that STING directly interacts with WIPI2, which is the key protein for LC3 lipidation in autophagy. Binding to WIPI2 is necessary for STING-induced autophagosome formation but does not affect STING activation and intracellular trafficking. In addition, the specific interaction between STING and the PI3P-binding motif of WIPI2 leads to the competition of WIPI2 binding between STING and PI3P, and mutual inhibition between STING-induced autophagy and canonical PI3P-dependent autophagy. Furthermore, we show that the STING-WIPI2 interaction is required for the clearance of cytoplasmic DNA and the attenuation of cGAS-STING signaling. Thus, the direct interaction between STING and WIPI2 enables STING to bypass the canonical upstream machinery to induce LC3 lipidation and autophagosome formation.
    Keywords:  STING; WIPI2; autophagy; cGAS; cytoplasmic DNA
    DOI:  https://doi.org/10.15252/embj.2022112387
  13. Mol Biol Cell. 2023 Mar 08. mbcE22080355
    BAG6 supports stress fiber formation by preventing the ubiquitin-mediated degradation of RhoA.
    Maho Miyauchi, Reina Matsumura, Hiroyuki Kawahara.
      The Rho family of small GTPases are key regulators of cytoskeletal actin polymerization. Although the ubiquitination of Rho proteins is reported to control their activity, the mechanisms by which the ubiquitination of Rho family proteins is controlled by ubiquitin ligases have yet to be elucidated. In this study, we identified BAG6 as the first factor needed to prevent the ubiquitination of RhoA, a critical Rho family protein in F-actin polymerization. We found that BAG6 is necessary for stress fiber formation by stabilizing endogenous RhoA. BAG6 deficiency enhanced the association between RhoA and Cullin-3-based ubiquitin ligases, thus promoting its polyubiquitination and subsequent degradation, leading to the abrogation of actin polymerization. In contrast, the restoration of RhoA expression through transient overexpression rescued the stress fiber formation defects induced by BAG6 depletion. BAG6 was also necessary for the appropriate assembly of focal adhesions as well as cell migration events. These findings reveal a novel role for BAG6 in maintaining the integrity of actin fiber polymerization and establish BAG6 as a RhoA-stabilizing holdase, which binds to and supports the function of RhoA. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-08-0355
  14. J Am Chem Soc. 2023 Mar 10.
    Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.
    Reina Iwase, Daniel R Dempsey, Samuel D Whedon, Hanjie Jiang, Brad A Palanski, Bedphiny Deng, Philip A Cole.
      Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.
    DOI:  https://doi.org/10.1021/jacs.2c13871
  15. Mol Syst Biol. 2023 Mar 10. e11024
    Phosphorylation-linked complex profiling identifies assemblies required for Hippo signal integration.
    Federico Uliana, Rodolfo Ciuffa, Ranjan Mishra, Andrea Fossati, Fabian Frommelt, Sabrina Keller, Martin Mehnert, Eivind Salmorin Birkeland, Frank van Drogen, Nevena Srejic, Matthias Peter, Nicolas Tapon, Ruedi Aebersold, Matthias Gstaiger.
      While several computational methods have been developed to predict the functional relevance of phosphorylation sites, experimental analysis of the interdependency between protein phosphorylation and Protein-Protein Interactions (PPIs) remains challenging. Here, we describe an experimental strategy to establish interdependencies between protein phosphorylation and complex formation. This strategy is based on three main steps: (i) systematically charting the phosphorylation landscape of a target protein; (ii) assigning distinct proteoforms of the target protein to different protein complexes by native complex separation (AP-BNPAGE) and protein correlation profiling; and (iii) analyzing proteoforms and complexes in cells lacking regulators of the target protein. We applied this strategy to YAP1, a transcriptional co-activator for the control of organ size and tissue homeostasis that is highly phosphorylated and among the most connected proteins in human cells. We identified multiple YAP1 phosphosites associated with distinct complexes and inferred how both are controlled by Hippo pathway members. We detected a PTPN14/LATS1/YAP1 complex and suggest a model how PTPN14 inhibits YAP1 via augmenting WW domain-dependent complex formation and phosphorylation by LATS1/2.
    Keywords:  Interaction proteomics; Protein phosphorylation; Protein-protein interactions; YAP1
    DOI:  https://doi.org/10.15252/msb.202211024
  16. J Cell Biol. 2023 Apr 03. pii: e202201027. [Epub ahead of print]222(4):
    The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA.
    Ellis G Jaffray, Michael H Tatham, Barbara Mojsa, Magda Liczmanska, Alejandro Rojas-Fernandez, Yili Yin, Graeme Ball, Ronald T Hay.
      Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.
    DOI:  https://doi.org/10.1083/jcb.202201027
  17. J Biol Chem. 2023 Mar 07. pii: S0021-9258(23)00235-1. [Epub ahead of print] 104593
    Differential role for rapid proteostasis in Rho GTPase-mediated control of quiescent endothelial integrity.
    Fabienne Podieh, Roos Wensveen, MaxC Overboom, Lotte Abbas, Jisca Majolée, PeterL Hordijk.
      Endothelial monolayer permeability is regulated by actin dynamics and vesicular traffic. Recently, ubiquitination was also implicated in the integrity of quiescent endothelium, as it differentially controls the localization and stability of adhesion- and signaling proteins. However, the more general effect of fast protein turnover on endothelial integrity is not clear. Here, we found that inhibition of E1 ubiquitin ligases induces a rapid, reversible loss of integrity in quiescent, primary human endothelial monolayers, accompanied by increased F-actin stress fibers and the formation of intercellular gaps. Concomitantly, total protein and activity of the actin-regulating GTPase RhoB, but not its close homologue RhoA, increase ∼10-fold in 5-8 h. We determined that, the depletion of RhoB, but not of RhoA, the inhibition of actin contractility and the inhibition of protein synthesis all significantly rescue the loss of cell-cell contact induced by E1 ligase inhibition. Collectively, our data suggest that in quiescent human endothelial cells, the continuous and fast turnover of short-lived proteins that negatively regulate cell-cell contact, is essential to preserve monolayer integrity.
    Keywords:  Rho GTPases; endothelial cells; monolayer integrity; proteostasis; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2023.104593
  18. J Integr Plant Biol. 2023 Mar 10.
    Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria.
    Junho Lee, Byeongho Moon, Dong Wook Lee, Inhwan Hwang.
      Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery. However, the situation is confusing for transmembrane domain (TMD)-containing signal anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs; a basic residue and an arginine-rich region flanking the N- and C-termini of TMDs, respectively, and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co-translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a posttranslational manner. This article is protected by copyright. All rights reserved.
    Keywords:  ER targeting; TMD; Translational slowdown; arginine-rich region; mitochondria targeting; organelle targeting; transmembrane domain
    DOI:  https://doi.org/10.1111/jipb.13475
  19. J Cell Biol. 2023 May 01. pii: e202205117. [Epub ahead of print]222(5):
    PP6 regulation of Aurora A-TPX2 limits NDC80 phosphorylation and mitotic spindle size.
    Tomoaki Sobajima, Katarzyna M Kowalczyk, Stefanos Skylakakis, Daniel Hayward, Luke J Fulcher, Colette Neary, Caleb Batley, Samvid Kurlekar, Emile Roberts, Ulrike Gruneberg, Francis A Barr.
      Amplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the catalytic subunit of PP6, have amplified Aurora A activity, and as we show here, enlarged mitotic spindles which fail to hold chromosomes tightly together in anaphase, causing defective nuclear structure. Using functional genomics to shed light on the processes underpinning these changes, we discover synthetic lethality between PPP6C and the kinetochore protein NDC80. We find that NDC80 is phosphorylated on multiple N-terminal sites during spindle formation by Aurora A-TPX2, exclusively at checkpoint-silenced, microtubule-attached kinetochores. NDC80 phosphorylation persists until spindle disassembly in telophase, is increased in PPP6C knockout cells, and is Aurora B-independent. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C knockout cells. In regulating NDC80 phosphorylation by Aurora A-TPX2, PP6 plays an important role in mitotic spindle formation and size control and thus the fidelity of cell division.
    DOI:  https://doi.org/10.1083/jcb.202205117
  20. Cancer Res. 2023 Mar 07. pii: CAN-22-3739. [Epub ahead of print]
    The tumor suppressor functions of ubiquitin ligase KPC1: from cell cycle control to NF-κB regulator.
    Diana Gulei, Rareș Drula, Gabriel Ghiaur, Anca Dana Buzoianu, Yelena Kravtsova-Ivantsiv, Ciprian Tomuleasa, Aaron Ciechanover.
      The ubiquitin-proteasome system (UPS) is responsible for up to 90% of intracellular protein degradation. Alterations in UPS are extensively involved in the development and advancement of malignant pathologies. Thus, the components of the UPS can become potential targets for cancer therapeutics. KPC1 is an E3 ubiquitin ligase component of the UPS that regulates key pathways and processes in cancer. KPC1 sustains the ubiquitination of cytoplasmic p27, determining its elimination and transition between cell cycle phases. KPC1 also regulates NF-κB signaling by inducing ubiquitination of p105 to allow subsequent proteasomal processing to the functional form p50. It has been shown that the KPC1-p50 duo is reduced or absent in multiple malignancies and that therapeutic reinforcement of the functional axis can exhibit significant tumor suppressor activity. Here, we highlight the potential role of KPC1 as a tumor-suppressor by fully describing its crucial role in p27 signaling and the canonical NF-κB pathway.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-3739
  21. Proc Natl Acad Sci U S A. 2023 Mar 14. 120(11): e2215732120
    Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target.
    Junzhi Zhou, Xiaohe Ma, Xingrui He, Beiying Chen, Jiao Yuan, Zhemin Jin, Lijing Li, Zhiguo Wang, Qian Xiao, Yafei Cai, Yongkang Zou.
      Immunotherapy of PD-L1/PD-1 blockage elicited impressive clinical benefits for cancer treatment. However, the relative low response and therapy resistance highlight the need to better understand the molecular regulation of PD-L1 in tumors. Here, we report that PD-L1 is a target of UFMylation. UFMylation of PD-L1 destabilizes PD-L1 by synergizing its ubiquitination. Inhibition of PD-L1 UFMylation via silencing of UFL1 or Ubiquitin-fold modifier 1 (UFM1), or the defective UFMylation of PD-L1, stabilizes the PD-L1 in multiple human and murine cancer cells, and undermines antitumor immunity in vitro and mice, respectively. Clinically, UFL1 expression was decreased in multiple cancers and lower expression of UFL1 negatively correlated with the response of anti-PD1 therapy in melanoma patients. Moreover, we identified a covalent inhibitor of UFSP2 that promoted the UFMylation activity and contributed to the combination therapy with PD-1 blockade. Our findings identified a previously unrecognized regulator of PD-L1 and highlighted UFMylation as a potential therapeutic target.
    Keywords:  PD-L1; UFMylation; immune checkpoint; post-translational modification; tumor immunity
    DOI:  https://doi.org/10.1073/pnas.2215732120
  22. J Neurosci. 2023 Mar 09. pii: JN-RM-1604-22. [Epub ahead of print]
    Inhibition of nonsense-mediated decay induces nociceptive sensitization through activation of the integrated stress response.
    June Bryan de la Peña, Rebecca Chase, Nikesh Kunder, Patrick R Smith, Tzu-Fang Lou, Alexander Stanowick, Prarthana Suresh, Tarjani Shukla, Samuel E Butcher, Theodore Price, Zachary T Campbell.
      RNA stability is meticulously controlled. Here, we sought to determine if an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of roughly 10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murine dorsal root ganglion (DRG) sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons including ATF4. ATF4 is preferentially translated during the integrated stress response (ISR). This led us to ask if suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-alpha phosphorylation and reduced the abundance of the eIF2-alpha phosphatase constitutive repressor of eIF2-alpha phosphorylation (CReP). Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR.SIGNIFICANCE STATEMENT:Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called NMD. Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1604-22.2023
  23. J Mol Biol. 2023 Mar 08. pii: S0022-2836(23)00098-0. [Epub ahead of print] 168042
    An insect viral protein disrupts stress granule formation in mammalian cells.
    Jibin Sadasivan, Anastasia Hyrina, Rachel DaSilva, Eric Jan.
      Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation. The mechanism underlying this process is not fully understood. Here, we show that overexpression of wild-type CrPV-1A, but not the CrPV-1A(R146A) mutant protein, inhibits distinct SG assembly pathways in HeLa cells. CrPV-1A mediated SG inhibition is independent of the Argonaute-2 (Ago-2) binding domain and the E3 ubiquitin ligase recruitment domain. CrPV-1A expression leads to nuclear poly(A)+ RNA accumulation and is correlated with the localization of CrPV-1A to the nuclear periphery. Finally, we show that the overexpression of CrPV-1A blocks FUS and TDP-43 granules, which are pathological hallmarks of neurodegenerative diseases. We propose a model whereby CrPV-1A expression in mammalian cells blocks SG formation by depleting cytoplasmic mRNA scaffolds via mRNA export inhibition. CrPV-1A may provide a new molecular tool to disassemble RNA-protein aggregates and potentially uncouple SG functions.
    Keywords:  RNA; RNA transport; dicistrovirus; stress granules; virus
    DOI:  https://doi.org/10.1016/j.jmb.2023.168042
  24. Cell Rep. 2023 Mar 10. pii: S2211-1247(23)00232-2. [Epub ahead of print]42(3): 112221
    Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.
    Francesca Filippini, Sébastien Nola, Ahmed Zahraoui, Kevin Roger, Mansoore Esmaili, Ji Sun, José Wojnacki, Anaïs Vlieghe, Philippe Bun, Stéphanie Blanchon, Jean-Christophe Rain, Jean-Marc Taymans, Marie-Christine Chartier-Harlin, Chiara Guerrera, Thierry Galli.
      The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts with the v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7 knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7.
    Keywords:  CP: Cell biology; LRRK2; Parkinson’s disease; SNARE; VAMP4; VAMP7; VGF; biomarker; neuron; pro-peptides; secretion; secretomics
    DOI:  https://doi.org/10.1016/j.celrep.2023.112221
  25. Oncogene. 2023 Mar 07.
    CDK13 phosphorylates the translation machinery and promotes tumorigenic protein synthesis.
    Chao Wu, Ting Xie, Ying Guo, Donghai Wang, Min Qiu, Ruyi Han, Guoliang Qing, Kaiwei Liang, Hudan Liu.
      Cyclin-dependent kinase 13 (CDK13) has been suggested to phosphorylate RNA polymerase II and is involved in transcriptional activation. However, whether CDK13 catalyzes other protein substrates and how CDK13 contributes to tumorigenesis remain largely unclear. We here identify key translation machinery components, 4E-BP1 and eIF4B, as novel CDK13 substrates. CDK13 directly phosphorylates 4E-BP1 at Thr46 and eIF4B at Ser422; genetically or pharmacologically inhibiting CDK13 disrupts mRNA translation. Polysome profiling analysis shows that MYC oncoprotein synthesis strictly depends on CDK13-regulated translation in colorectal cancer (CRC), and CDK13 is required for CRC cell proliferation. As mTORC1 is implicated in 4E-BP1 and eIF4B phosphorylation, inactivation of CDK13 in combination with the mTORC1 inhibitor rapamycin further dephosphorylates 4E-BP1 and eIF4B and blocks protein synthesis. As a result, dual inhibition of CDK13 and mTORC1 induces more profound tumor cell death. These findings clarify the pro-tumorigenic role of CDK13 by direct phosphorylation of translation initiation factors and enhancing protein synthesis. Therefore, therapeutic targeting of CDK13 alone or in combination with rapamycin may pave a new way for cancer treatment.
    DOI:  https://doi.org/10.1038/s41388-023-02653-2
  26. Expert Opin Drug Discov. 2023 Mar 09. 1-17
    Novel approaches to targeted protein degradation technologies in drug discovery.
    Yu Xue, Andrew A Bolinger, Jia Zhou.
      INTRODUCTION: Target protein degradation (TPD) provides a novel therapeutic modality, other than inhibition, through the direct depletion of target proteins. Two primary human protein homeostasis mechanisms are exploited: the ubiquitin-proteasome system (UPS) and the lysosomal system. TPD technologies based on these two systems are progressing at an impressive pace.AREAS COVERED: This review focuses on the TPD strategies based on UPS and lysosomal system, mainly classified into three types: Molecular Glue (MG), PROteolysis Targeting Chimera (PROTAC), and lysosome-mediated TPD. Starting with a brief background introduction of each strategy, exciting examples and perspectives on these novel approaches are provided.
    EXPERT OPINION: MGs and PROTACs are two major UPS-based TPD strategies that have been extensively investigated in the past decade. Despite some clinical trials, several critical issues remain, among which is emphasized by the limitation of targets. Recently developed lysosomal system-based approaches provide alternative solutions for TPD beyond UPS' capability. The newly emerging novel approaches may partially address issues that have long plagued researchers, such as low potency, poor cell permeability, on-/off-target toxicity, and delivery efficiency. Comprehensive considerations for the rational design of protein degraders and continuous efforts to seek effective solutions are imperative to advance these strategies into clinical medications.
    Keywords:  AUTOphagy-TArgeting Chimera (AUTOTAC); AUtophagy-TArgeting Chimera (AUTAC); AuTophagosome-TEthering Compound (ATTEC); LYsosome-TArgeting Chimera (LYTAC); Molecular Glue (MG); PROteolysis Targeting Chimera (PROTAC); Target Protein Degradation (TPD); Ubiquitin-Proteasome System (UPS); drug discovery; lysosomal system; protein degraders
    DOI:  https://doi.org/10.1080/17460441.2023.2187777
  27. J Biol Chem. 2023 Mar 03. pii: S0021-9258(23)00224-7. [Epub ahead of print] 104582
    Coupling substrate-trapping with proximity-labeling to identify protein tyrosine phosphatase PTP1B signaling networks.
    ChristopherA Bonham, Vinay Mandati, RakeshK Singh, DarrylJ Pappin, NicholasK Tonks.
      The ability to define functional interactions between enzymes and their substrates is crucial for understanding biological control mechanisms; however, such methods face challenges in the transient nature and low stoichiometry of enzyme-substrate interactions. Now, we have developed an optimized strategy that couples substrate-trapping mutagenesis to proximity-labeling mass spectrometry for quantitative analysis of protein complexes involving the protein tyrosine phosphatase PTP1B. This methodology represents a significant shift from classical schemes; it is capable of being performed at near-endogenous expression levels and increasing stoichiometry of target enrichment without a requirement for stimulation of supraphysiological tyrosine phosphorylation levels or maintenance of substrate complexes during lysis and enrichment procedures. Advantages of this new approach are illustrated through application to PTP1B interaction networks in models of HER2-positive and Herceptin-resistant breast cancer. We have demonstrated that inhibitors of PTP1B significantly reduced proliferation and viability in cell-based models of acquired and de novo Herceptin resistance in HER2-positive breast cancer. Using differential analysis, comparing substrate-trapping to wild-type PTP1B, we have identified multiple unreported protein targets of PTP1B with established links to HER2-induced signaling and provided internal validation of method specificity through overlap with previously identified substrate candidates. Overall, this versatile approach can be readily integrated with evolving proximity-labeling platforms (TurboID, BioID2, etc.), and is broadly applicable across all PTP family members for the identification of conditional substrate specificities and signaling nodes in models of human disease.
    Keywords:  chemical biology; mass spectrometry; protein tyrosine phosphatase; protein-protein interaction; proximity-labeling; signal transduction; substrate-trapping; tyrosine phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2023.104582
  28. J Med Chem. 2023 Mar 09.
    ARE-PROTACs Enable Co-degradation of an Nrf2-MafG Heterodimer.
    Jianai Ji, Sinan Ma, Yuxuan Zhu, Jinglong Zhao, Yuanyuan Tong, Qidong You, Zhengyu Jiang.
      Proteolysis-targeting chimera (PROTAC) technology has emerged as a potential strategy to degrade "undruggable" proteins in recent years. Nrf2, an aberrantly activated transcription factor in cancer, is generally considered undruggable as lacking active sites or allosteric pockets. Here, we constructed the chimeric molecule C2, which consists of an Nrf2-binding element and a CRBN ligand, as a first-in-class Nrf2 degrader. Surprisingly, C2 was found to selectively degrade an Nrf2-MafG heterodimer simultaneously via the ubiquitin-proteasome system. C2 impeded Nrf2-ARE transcriptional activity significantly and improved the sensitivity of NSCLC cells to ferroptosis and therapeutic drugs. The degradation character of ARE-PROTACs suggests that the PROTAC hijacking the transcription element of TFs could achieve co-degradation of the transcription complex.
    DOI:  https://doi.org/10.1021/acs.jmedchem.2c01909
  29. iScience. 2023 Mar 17. 26(3): 106094
    Identifying multiscale translational safety biomarkers using a network-based systems approach.
    Giulia Callegaro, Johannes P Schimming, Janet Piñero González, Steven J Kunnen, Lukas Wijaya, Panuwat Trairatphisan, Linda van den Berk, Kim Beetsma, Laura I Furlong, Jeffrey J Sutherland, Jennifer Mollon, James L Stevens, Bob van de Water.
      Animal testing is the current standard for drug and chemicals safety assessment, but hazards translation to human is uncertain. Human in vitro models can address the species translation but might not replicate in vivo complexity. Herein, we propose a network-based method addressing these translational multiscale problems that derives in vivo liver injury biomarkers applicable to in vitro human early safety screening. We applied weighted correlation network analysis (WGCNA) to a large rat liver transcriptomic dataset to obtain co-regulated gene clusters (modules). We identified modules statistically associated with liver pathologies, including a module enriched for ATF4-regulated genes as associated with the occurrence of hepatocellular single-cell necrosis, and as preserved in human liver in vitro models. Within the module, we identified TRIB3 and MTHFD2 as a novel candidate stress biomarkers, and developed and used BAC-eGFPHepG2 reporters in a compound screening, identifying compounds showing ATF4-dependent stress response and potential early safety signals.
    Keywords:  Bioinformatics; Gene network; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2023.106094
  30. Science. 2023 Mar 10. 379(6636): 996-1003
    Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.
    Kevin G Hicks, Ahmad A Cluntun, Heidi L Schubert, Sean R Hackett, Jordan A Berg, Paul G Leonard, Mariana A Ajalla Aleixo, Youjia Zhou, Alex J Bott, Sonia R Salvatore, Fei Chang, Aubrie Blevins, Paige Barta, Samantha Tilley, Aaron Leifer, Andrea Guzman, Ajak Arok, Sarah Fogarty, Jacob M Winter, Hee-Chul Ahn, Karen N Allen, Samuel Block, Iara A Cardoso, Jianping Ding, Ingrid Dreveny, William C Gasper, Quinn Ho, Atsushi Matsuura, Michael J Palladino, Sabin Prajapati, Pengkai Sun, Kai Tittmann, Dean R Tolan, Judith Unterlass, Andrew P VanDemark, Matthew G Vander Heiden, Bradley A Webb, Cai-Hong Yun, Pengkai Zhao, Bei Wang, Francisco J Schopfer, Christopher P Hill, Maria Cristina Nonato, Florian L Muller, James E Cox, Jared Rutter.
      Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.
    DOI:  https://doi.org/10.1126/science.abm3452
  31. RNA. 2023 Mar 06. pii: rna.079525.122. [Epub ahead of print]
    Translation re-initiation after uORFs does not fully protect mRNAs from nonsense-mediated decay.
    Paul J Russell, Jacob A Slivka, Elaina P Boyle, Arthur H M Burghes, Michael G Kearse.
      It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5' UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability in HeLa cells. Using custom 5' UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives in HeLa cells and mining available mRNA half-life datasets for cumulative predicted uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to prevent NMD. Together, these data suggests that the decision of whether NMD ensues after translating uORFs occurs before re-initiation in mammalian cells.
    Keywords:  NMD; eIF; mRNA decay; ribosome; translational control
    DOI:  https://doi.org/10.1261/rna.079525.122
  32. Commun Biol. 2023 Mar 09. 6(1): 252
    Sustained overexpression of spliced X-box-binding protein-1 in neurons leads to spontaneous seizures and sudden death in mice.
    Zhuoran Wang, Qiang Li, Brad J Kolls, Brian Mace, Shu Yu, Xuan Li, Wei Liu, Eduardo Chaparro, Yuntian Shen, Lihong Dang, Ángela Del Águila, Joshua D Bernstock, Kory R Johnson, Junjie Yao, William C Wetsel, Scott D Moore, Dennis A Turner, Wei Yang.
      The underlying etiologies of seizures are highly heterogeneous and remain incompletely understood. While studying the unfolded protein response (UPR) pathways in the brain, we unexpectedly discovered that transgenic mice (XBP1s-TG) expressing spliced X-box-binding protein-1 (Xbp1s), a key effector of UPR signaling, in forebrain excitatory neurons, rapidly develop neurologic deficits, most notably recurrent spontaneous seizures. This seizure phenotype begins around 8 days after Xbp1s transgene expression is induced in XBP1s-TG mice, and by approximately 14 days post induction, the seizures evolve into status epilepticus with nearly continuous seizure activity followed by sudden death. Animal death is likely due to severe seizures because the anticonvulsant valproic acid could significantly prolong the lives of XBP1s-TG mice. Mechanistically, our gene profiling analysis indicates that compared to control mice, XBP1s-TG mice exhibit 591 differentially regulated genes (mostly upregulated) in the brain, including several GABAA receptor genes that are notably downregulated. Finally, whole-cell patch clamp analysis reveals a significant reduction in both spontaneous and tonic GABAergic inhibitory responses in Xbp1s-expressing neurons. Taken together, our findings unravel a link between XBP1s signaling and seizure occurrence.
    DOI:  https://doi.org/10.1038/s42003-023-04594-8
  33. Proc Natl Acad Sci U S A. 2023 Mar 14. 120(11): e2214556120
    Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains.
    Jing Yang John Wang, Alena Khmelinskaia, William Sheffler, Marcos C Miranda, Aleksandar Antanasijevic, Andrew J Borst, Susana V Torres, Chelsea Shu, Yang Hsia, Una Nattermann, Daniel Ellis, Carl Walkey, Maggie Ahlrichs, Sidney Chan, Alex Kang, Hannah Nguyen, Claire Sydeman, Banumathi Sankaran, Mengyu Wu, Asim K Bera, Lauren Carter, Brooke Fiala, Michael Murphy, David Baker, Andrew B Ward, Neil P King.
      Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.
    Keywords:  biochemistry; nanoparticles; protein design
    DOI:  https://doi.org/10.1073/pnas.2214556120
  34. Proc Natl Acad Sci U S A. 2023 Mar 14. 120(11): e2207974120
    De novo design of small beta barrel proteins.
    David E Kim, Davin R Jensen, David Feldman, Doug Tischer, Ayesha Saleem, Cameron M Chow, Xinting Li, Lauren Carter, Lukas Milles, Hannah Nguyen, Alex Kang, Asim K Bera, Francis C Peterson, Brian F Volkman, Sergey Ovchinnikov, David Baker.
      Small beta barrel proteins are attractive targets for computational design because of their considerable functional diversity despite their very small size (<70 amino acids). However, there are considerable challenges to designing such structures, and there has been little success thus far. Because of the small size, the hydrophobic core stabilizing the fold is necessarily very small, and the conformational strain of barrel closure can oppose folding; also intermolecular aggregation through free beta strand edges can compete with proper monomer folding. Here, we explore the de novo design of small beta barrel topologies using both Rosetta energy-based methods and deep learning approaches to design four small beta barrel folds: Src homology 3 (SH3) and oligonucleotide/oligosaccharide-binding (OB) topologies found in nature and five and six up-and-down-stranded barrels rarely if ever seen in nature. Both approaches yielded successful designs with high thermal stability and experimentally determined structures with less than 2.4 Å rmsd from the designed models. Using deep learning for backbone generation and Rosetta for sequence design yielded higher design success rates and increased structural diversity than Rosetta alone. The ability to design a large and structurally diverse set of small beta barrel proteins greatly increases the protein shape space available for designing binders to protein targets of interest.
    Keywords:  high-throughput screening; machine learning; protein design; small beta barrels
    DOI:  https://doi.org/10.1073/pnas.2207974120
  35. Cell Host Microbe. 2023 Mar 08. pii: S1931-3128(23)00075-6. [Epub ahead of print]31(3): 327-328
    Stressing out over mucus secretion.
    Michael J Grey.
      In this issue of Cell Host & Microbe, Naama et al. show that autophagy controls mucus secretion in the colons of mice. They demonstrate that autophagy reduces ER stress in mucus-producing goblet cells to enhance mucus production, which shapes the gut microbial community and protects against colitis.
    DOI:  https://doi.org/10.1016/j.chom.2023.02.005
  36. Cell Rep. 2023 Mar 07. pii: S2211-1247(23)00239-5. [Epub ahead of print]42(3): 112228
    RNA-binding protein FXR1 drives cMYC translation by recruiting eIF4F complex to the translation start site.
    Jasmine George, Yongsheng Li, Ishaque P Kadamberi, Deepak Parashar, Shirng-Wern Tsaih, Prachi Gupta, Anjali Geethadevi, Changliang Chen, Chandrima Ghosh, Yunguang Sun, Sonam Mittal, Ramani Ramchandran, Hallgeir Rui, Gabriel Lopez-Berestein, Cristian Rodriguez-Aguayo, Gustavo Leone, Janet S Rader, Anil K Sood, Madhusudan Dey, Sunila Pradeep, Pradeep Chaluvally-Raghavan.
      
    DOI:  https://doi.org/10.1016/j.celrep.2023.112228
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