bims-scepro Biomed News
on Stem cell proteostasis
Issue of 2024–02–25
twenty papers selected by
William Grey, University of York
  1. Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo.
  2. Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate.
  3. Lysine Methylation-Dependent Proteolysis by the Malignant Brain Tumor (MBT) Domain Proteins.
  4. CD38-directed, single-chain T cell-engager targets leukemia stem cells through IFNγ-induced CD38 expression.
  5. Mitochondrial Dysfunction and Protein Homeostasis in Aging: Insights from a Premature-Aging Mouse Model.
  6. N-recognins UBR1 and UBR2 as central ER stress sensors in mammals.
  7. Using stem cells to model the human bone marrow in a dish.
  8. Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution.
  9. Serine mistranslation induces the integrated stress response without accumulation of uncharged tRNAs.
  10. Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability.
  11. TFEB activation triggers pexophagy for functional adaptation during oxidative stress under calcium deficient-conditions.
  12. E3 Ligases Regulate Organelle Inheritance in Yeast.
  13. HRD1-induced TMEM2 ubiquitination promotes ER stress-mediated apoptosis through a non-canonical pathway in intestinal ischemia/reperfusion.
  14. Targeted protein degradation via intramolecular bivalent glues.
  15. Generation of complex bone marrow organoids from human induced pluripotent stem cells.
  16. Nutrient metabolism in regulating intestinal stem cell homeostasis.
  17. mTORC1 activity oscillates throughout the cell cycle promoting mitotic entry and differentially influencing autophagy induction.
  18. Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.
  19. Ageing-dependent thiol oxidation reveals early oxidation of proteins with core proteostasis functions.
  20. Targeted protein degradation directly engaging lysosomes or proteasomes.
  1. Nat Commun. 2024 Feb 23. 15(1): 1653
    Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo.
    Zaniah N Gonzalez Galofre, Alastair M Kilpatrick, Madalena Marques, Diana Sá da Bandeira, Telma Ventura, Mario Gomez Salazar, Léa Bouilleau, Yvan Marc, Ana B Barbosa, Fiona Rossi, Mariana Beltran, Harmen J G van de Werken, Wilfred F J van IJcken, Neil C Henderson, Stuart J Forbes, Mihaela Crisan.
      Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway, suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however, their role in the hematopoietic development in vivo remains unknown. Here, we identify a subpopulation of NG2+Runx1+ perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2+ cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs, endothelial cells and hematopoietic cells in the murine AGM. Importantly, this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental, as NG2+Runx1+ cells were not detected in the adult bone marrow, demonstrating the existence of a specialised pericyte population in the HSC-generating niche, unique to the embryo.
    DOI:  https://doi.org/10.1038/s41467-024-44913-z
  2. Nat Commun. 2024 Feb 21. 15(1): 1604
    Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate.
    Roshana Thambyrajah, Maria Maqueda, Wen Hao Neo, Kathleen Imbach, Yolanda Guillén, Daniela Grases, Zaki Fadlullah, Stefano Gambera, Francesca Matteini, Xiaonan Wang, Fernando J Calero-Nieto, Manel Esteller, Maria Carolina Florian, Eduard Porta, Rui Benedito, Berthold Göttgens, Georges Lacaud, Lluis Espinosa, Anna Bigas.
      Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.
    DOI:  https://doi.org/10.1038/s41467-024-45716-y
  3. Int J Mol Sci. 2024 Feb 13. pii: 2248. [Epub ahead of print]25(4):
    Lysine Methylation-Dependent Proteolysis by the Malignant Brain Tumor (MBT) Domain Proteins.
    Hong Sun, Hui Zhang.
      Lysine methylation is a major post-translational protein modification that occurs in both histones and non-histone proteins. Emerging studies show that the methylated lysine residues in non-histone proteins provide a proteolytic signal for ubiquitin-dependent proteolysis. The SET7 (SETD7) methyltransferase specifically transfers a methyl group from S-Adenosyl methionine to a specific lysine residue located in a methylation degron motif of a protein substrate to mark the methylated protein for ubiquitin-dependent proteolysis. LSD1 (Kdm1a) serves as a demethylase to dynamically remove the methyl group from the modified protein. The methylated lysine residue is specifically recognized by L3MBTL3, a methyl-lysine reader that contains the malignant brain tumor domain, to target the methylated proteins for proteolysis by the CRL4DCAF5 ubiquitin ligase complex. The methylated lysine residues are also recognized by PHF20L1 to protect the methylated proteins from proteolysis. The lysine methylation-mediated proteolysis regulates embryonic development, maintains pluripotency and self-renewal of embryonic stem cells and other stem cells such as neural stem cells and hematopoietic stem cells, and controls other biological processes. Dysregulation of the lysine methylation-dependent proteolysis is associated with various diseases, including cancers. Characterization of lysine methylation should reveal novel insights into how development and related diseases are regulated.
    Keywords:  CRL4; DCAF5; L3MBTL3; LSD1/Kdm1a; SET7/SETD7; demethylation; lysine methylation; ubiquitin E3 ligase
    DOI:  https://doi.org/10.3390/ijms25042248
  4. Blood. 2024 Feb 26. pii: blood.2023021570. [Epub ahead of print]
    CD38-directed, single-chain T cell-engager targets leukemia stem cells through IFNγ-induced CD38 expression.
    Mariam Murtadha, Miso Park, Yinghui Zhu, Enrico Caserta, Ottavio Napolitano, Theophilus Tandoh, Milad Moloudizargari, Alex Pozhitkov, Mahmoud K Singer, Ada Alice Dona, Hawa Vahed, Asaul Gonzalez, Kevin Ly, Ching Ouyang, James Sanchez, Lokesh Nigam, Amanda Duplan, Arnab Chowdhury, Lucy Y Ghoda, Ling Li, Bin Zhang, Amrita Y Krishnan, Guido Marcucci, John C Williams, Flavia Pichiorri.
      Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that IFNγ reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFNγ-induced CD38 upregulation depends on IRF-1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T cell engager (BN-CD38) designed to promote an effective immunological synapse between both CD8 and CD4 T cells and CD38pos AML cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts leading to T cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFNγ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, while BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.
    DOI:  https://doi.org/10.1182/blood.2023021570
  5. Biomolecules. 2024 Jan 30. pii: 162. [Epub ahead of print]14(2):
    Mitochondrial Dysfunction and Protein Homeostasis in Aging: Insights from a Premature-Aging Mouse Model.
    Jaime M Ross, Lars Olson, Giuseppe Coppotelli.
      Mitochondrial dysfunction has been implicated in aging and age-related disorders. Disturbed-protein homeostasis and clearance of damaged proteins have also been linked to aging, as well as to neurodegenerative diseases, cancers, and metabolic disorders. However, since mitochondrial oxidative phosphorylation, ubiquitin-proteasome, and autophagy-lysosome systems are tightly interdependent, it is not understood whether the facets observed in aging are the causes or consequences of one or all of these failed processes. We therefore used prematurely aging mtDNA-mutator mice and normally aging wild-type littermates to elucidate whether mitochondrial dysfunction per se is sufficient to impair cellular protein homeostasis similarly to that which is observed in aging. We found that both mitochondrial dysfunction and normal aging affect the ubiquitin-proteasome system in a tissue-dependent manner, whereas only normal aging markedly impairs the autophagy-lysosome system. Thus, our data show that the proteostasis network control in the prematurely aging mtDNA-mutator mouse differs in certain aspects from that found in normal aging. Taken together, our findings suggest that severe mitochondrial dysfunction drives an aging phenotype associated with the impairment of certain components of the protein homeostasis machinery, while others, such as the autophagy-lysosome system, are not affected or only minimally affected. Taken together, this shows that aging is a multifactorial process resulting from alterations of several integrated biological processes; thus, manipulating one process at the time might not be sufficient to fully recapitulate all changes associated with normal aging.
    Keywords:  aging; autophagy; mitochondrial dysfunction; ubiquitin–proteasome system
    DOI:  https://doi.org/10.3390/biom14020162
  6. Mol Cells. 2024 Jan;pii: S1016-8478(23)25251-6. [Epub ahead of print]47(1): 100001
    N-recognins UBR1 and UBR2 as central ER stress sensors in mammals.
    Ly Thi Huong Luu Le, Seoyoung Park, Jung Hoon Lee, Yun Kyung Kim, Min Jae Lee.
      In eukaryotes, a primary protein quality control (PQC) process involves the destruction of conformationally misfolded proteins through the ubiquitin-proteasome system. Because approximately one-third of eukaryotic proteomes fold and assemble within the endoplasmic reticulum (ER) before being sent to their destinations, the ER plays a crucial role in PQC. The specific functions and biochemical roles of several E3 ubiquitin ligases involved in ER-associated degradation in mammals, on the other hand, are mainly unknown. We identified 2 E3 ligases, ubiquitin protein ligase E3 component N-recognin 1 (UBR1) and ubiquitin protein ligase E3 component N-recognin 2 (UBR2), which are the key N-recognins in the N-degron pathway and participate in the ER stress response in mammalian cells by modulating their stability. Cells lacking UBR1 and UBR2 are hypersensitive to ER stress-induced apoptosis. Under normal circumstances, these proteins are polyubiquitinated through Lys48-specific linkages and are then degraded by the 26S proteasome. In contrast, when cells are subjected to ER stress, UBR1 and UBR2 exhibit greater stability, potentially as a cellular adaptive response to stressful conditions. Although the precise mechanisms underlying these findings require further investigation, our findings show that cytoplasmic UBR1 and UBR2 have anti-ER stress activities and contribute to global PQC in mammals. These data also reveal an additional level of complexity within the mammalian ER-associated degradation system, implicating potential involvement of the N-degron pathway.
    Keywords:  Auto-ubiquitination; Endoplasmic reticulum stress; N-degron pathway; Protein quality control; Thapsigargin
    DOI:  https://doi.org/10.1016/j.mocell.2023.12.001
  7. Nat Methods. 2024 Feb 19.
    Using stem cells to model the human bone marrow in a dish.
      
    DOI:  https://doi.org/10.1038/s41592-024-02173-1
  8. Cell Death Dis. 2024 Feb 21. 15(2): 159
    Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution.
    Seyram Yao Adzraku, Can Cao, Qi Zhou, Ke Yuan, Xiaowen Hao, Yue Li, Shengnan Yuan, Yujin Huang, Kailin Xu, Jianlin Qiao, Wen Ju, Lingyu Zeng.
      Bone marrow ablation is routinely performed before hematopoietic stem cell transplantation (HSCT). Hematopoietic stem and progenitor cells (HSPCs) require a stable bone marrow microenvironment to expand and refill the peripheral blood cell pool after ablation. Roundabout guidance receptor 4 (Robo4) is a transmembrane protein exclusive to endothelial cells and is vital in preserving vascular integrity. Hence, the hypothesis is that Robo4 maintains the integrity of bone marrow endothelial cells following radiotherapy. We created an endothelial cell injury model with γ-radiation before Robo4 gene manipulation using lentiviral-mediated RNAi and gene overexpression techniques. We demonstrate that Robo4 and specific mesenchymal proteins (Fibronectin, Vimentin, αSma, and S100A4) are upregulated in endothelial cells exposed to irradiation (IR). We found that Robo4 depletion increases the expression of endoglin (CD105), an auxiliary receptor for the transforming growth factor (TGF-β) family of proteins, and promotes endothelial-to-mesenchymal transition (End-MT) through activation of both the canonical (Smad) and non-canonical (AKT/NF-κB) signaling pathways to facilitate Snail1 activation and its nuclear translocation. Endothelial Robo4 overexpression stimulates the expression of immunoglobulin-like adhesion molecules (ICAM-1 and VCAM-1) and alleviates irradiation-induced End-MT. Our coculture model showed that transcriptional downregulation of endothelial Robo4 reduces HSPC proliferation and increases HSC quiescence and apoptosis. However, Robo4 overexpression mitigated the damaged endothelium's suppressive effects on HSC proliferation and differentiation. These findings indicate that by controlling End-MT, Robo4 preserves microvascular integrity after radiation preconditioning, protects endothelial function, and lessens the inhibitory effect of damaged endothelium on hematopoietic reconstitution.
    DOI:  https://doi.org/10.1038/s41419-024-06546-4
  9. bioRxiv. 2024 Feb 08. pii: 2024.02.04.578812. [Epub ahead of print]
    Serine mistranslation induces the integrated stress response without accumulation of uncharged tRNAs.
    Hong Zhang, Jiqiang Ling.
      Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that support robust and accurate protein synthesis. A rapidly expanding number of studies show that mutations in aaRSs lead to multiple human diseases, including neurological disorders and cancer. Much remains unknown about how aaRS mutations impact human health. In particular, how aminoacylation errors affect stress responses and fitness in eukaryotic cells remains poorly understood. The integrated stress response (ISR) is an adaptive mechanism in response to multiple stresses. However, chronic activation of the ISR contributes to the development of multiple diseases (e.g., neuropathies). Here we show that Ser misincorporation into Ala and Thr codons, resulting from aaRS editing defects or mutations in tRNAs, constitutively active the ISR. Such activation does not appear to depend on the accumulation of uncharged tRNAs, implicating that Ser mistranslation may lead to ribosome stalling and collision.
    DOI:  https://doi.org/10.1101/2024.02.04.578812
  10. Proc Natl Acad Sci U S A. 2024 Feb 27. 121(9): e2322582121
    Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability.
    Undiagnosed Diseases Network
      Nascent proteins destined for the cell membrane and the secretory pathway are targeted to the endoplasmic reticulum (ER) either posttranslationally or cotranslationally. The signal-independent pathway, containing the protein TMEM208, is one of three pathways that facilitates the translocation of nascent proteins into the ER. The in vivo function of this protein is ill characterized in multicellular organisms. Here, we generated a CRISPR-induced null allele of the fruit fly ortholog CG8320/Tmem208 by replacing the gene with the Kozak-GAL4 sequence. We show that Tmem208 is broadly expressed in flies and that its loss causes lethality, although a few short-lived flies eclose. These animals exhibit wing and eye developmental defects consistent with impaired cell polarity and display mild ER stress. Tmem208 physically interacts with Frizzled (Fz), a planar cell polarity (PCP) receptor, and is required to maintain proper levels of Fz. Moreover, we identified a child with compound heterozygous variants in TMEM208 who presents with developmental delay, skeletal abnormalities, multiple hair whorls, cardiac, and neurological issues, symptoms that are associated with PCP defects in mice and humans. Additionally, fibroblasts of the proband display mild ER stress. Expression of the reference human TMEM208 in flies fully rescues the loss of Tmem208, and the two proband-specific variants fail to rescue, suggesting that they are loss-of-function alleles. In summary, our study uncovers a role of TMEM208 in development, shedding light on its significance in ER homeostasis and cell polarity.
    Keywords:  Drosophila; ER stress; PCP; TMEM proteins; hSnd2
    DOI:  https://doi.org/10.1073/pnas.2322582121
  11. Cell Commun Signal. 2024 Feb 21. 22(1): 142
    TFEB activation triggers pexophagy for functional adaptation during oxidative stress under calcium deficient-conditions.
    Laxman Manandhar, Raghbendra Kumar Dutta, Pradeep Devkota, Arun Chhetri, Xiaofan Wei, Channy Park, Hyug Moo Kwon, Raekil Park.
       BACKGROUND: Calcium is a ubiquitous intracellular messenger that regulates the expression of various genes involved in cell proliferation, differentiation, and motility. The involvement of calcium in diverse metabolic pathways has been suggested. However, the effect of calcium in peroxisomes, which are involved in fatty acid oxidation and scavenges the result reactive oxygen species (ROS), remains elusive. In addition, impaired peroxisomal ROS inhibit the mammalian target of rapamycin complex 1 (mTORC1) and promote autophagy. Under stress, autophagy serves as a protective mechanism to avoid cell death. In response to oxidative stress, lysosomal calcium mediates transcription factor EB (TFEB) activation. However, the impact of calcium on peroxisome function and the mechanisms governing cellular homeostasis to prevent diseases caused by calcium deficiency are currently unknown.
    METHODS: To investigate the significance of calcium in peroxisomes and their roles in preserving cellular homeostasis, we established an in-vitro scenario of calcium depletion.
    RESULTS: This study demonstrated that calcium deficiency reduces catalase activity, resulting in increased ROS accumulation in peroxisomes. This, in turn, inhibits mTORC1 and induces pexophagy through TFEB activation. However, treatment with the antioxidant N-acetyl-l-cysteine (NAC) and the autophagy inhibitor chloroquine impeded the nuclear translocation of TFEB and attenuated peroxisome degradation.
    CONCLUSIONS: Collectively, our study revealed that ROS-mediated TFEB activation triggers pexophagy during calcium deficiency, primarily because of attenuated catalase activity. We posit that calcium plays a significant role in the proper functioning of peroxisomes, critical for fatty-acid oxidation and ROS scavenging in maintaining cellular homeostasis. These findings have important implications for signaling mechanisms in various pathologies, including Zellweger's syndrome and ageing.
    Keywords:  Autophagy; Calcium; Catalase; Peroxisome; ROS; TFEB
    DOI:  https://doi.org/10.1186/s12964-024-01524-x
  12. Cells. 2024 Feb 06. pii: 292. [Epub ahead of print]13(4):
    E3 Ligases Regulate Organelle Inheritance in Yeast.
    Keisuke Obara, Kohei Nishimura, Takumi Kamura.
      Saccharomyces cerevisiae proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin-myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.
    Keywords:  E3 ligase; mitochondria; myosin; peroxisomes; proteolysis; ubiquitin; vacuoles; yeast
    DOI:  https://doi.org/10.3390/cells13040292
  13. Cell Death Dis. 2024 Feb 20. 15(2): 154
    HRD1-induced TMEM2 ubiquitination promotes ER stress-mediated apoptosis through a non-canonical pathway in intestinal ischemia/reperfusion.
    Xuzi Zhao, Deshun Liu, Yan Zhao, Zhecheng Wang, Yue Wang, Zhao Chen, Shili Ning, Guangzhi Wang, Lu Meng, Jihong Yao, Xiaofeng Tian.
      Intestinal ischemia/reperfusion (I/R) injury is a typical pathological course in the clinic with a high morbidity rate. Recent research has pointed out the critical role of ubiquitination during the occurrence and development of intestinal I/R by precisely mediating protein quality control and function. Here, we conducted an integrated multiomic analysis to identify critical ubiquitination-associated molecules in intestinal I/R and identified endoplasmic reticulum-located HRD1 as a candidate molecule. During intestinal I/R, excessive ER stress plays a central role by causing apoptotic pathway activation. In particular, we found that ER stress-mediated apoptosis was mitigated by HRD1 knockdown in intestinal I/R mice. Mechanistically, TMEM2 was identified as a new substrate of HRD1 in intestinal I/R by mass spectrometry analysis, which has a crucial role in attenuating apoptosis and promoting non-canonical ER stress resistance. A strong negative correlation was found between the protein levels of HRD1 and TMEM2 in human intestinal ischemia samples. Specifically, HRD1 interacted with the lysine 42 residue of TMEM2 and reduced its stabilization by K48-linked polyubiquitination. Furthermore, KEGG pathway analysis revealed that TMEM2 regulated ER stress-mediated apoptosis in association with the PI3k/Akt signaling pathway rather than canonical ER stress pathways. In summary, HRD1 regulates ER stress-mediated apoptosis through a non-canonical pathway by ubiquitinating TMEM2 and inhibiting PI3k/Akt activation during intestinal I/R. The current study shows that HRD1 is an intestinal I/R critical regulator and that targeting the HRD1/TMEM2 axis may be a promising therapeutic approach.
    DOI:  https://doi.org/10.1038/s41419-024-06504-0
  14. Nature. 2024 Feb 21.
    Targeted protein degradation via intramolecular bivalent glues.
    Oliver Hsia, Matthias Hinterndorfer, Angus D Cowan, Kentaro Iso, Tasuku Ishida, Ramasubramanian Sundaramoorthy, Mark A Nakasone, Hana Imrichova, Caroline Schätz, Andrea Rukavina, Koraljka Husnjak, Martin Wegner, Alejandro Correa-Sáez, Conner Craigon, Ryan Casement, Chiara Maniaci, Andrea Testa, Manuel Kaulich, Ivan Dikic, Georg E Winter, Alessio Ciulli.
      Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.
    DOI:  https://doi.org/10.1038/s41586-024-07089-6
  15. Nat Methods. 2024 Feb 19.
    Generation of complex bone marrow organoids from human induced pluripotent stem cells.
    Stephanie Frenz-Wiessner, Savannah D Fairley, Maximilian Buser, Isabel Goek, Kirill Salewskij, Gustav Jonsson, David Illig, Benedicta Zu Putlitz, Daniel Petersheim, Yue Li, Pin-Hsuan Chen, Martina Kalauz, Raffaele Conca, Michael Sterr, Johanna Geuder, Yoko Mizoguchi, Remco T A Megens, Monika I Linder, Daniel Kotlarz, Martina Rudelius, Josef M Penninger, Carsten Marr, Christoph Klein.
      The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.
    DOI:  https://doi.org/10.1038/s41592-024-02172-2
  16. Cell Prolif. 2024 Feb 22. e13602
    Nutrient metabolism in regulating intestinal stem cell homeostasis.
    Ruicheng Shi, Bo Wang.
      Intestinal stem cells (ISCs) are known for their remarkable proliferative capacity, making them one of the most active cell populations in the body. However, a high turnover rate of intestinal epithelium raises the likelihood of dysregulated homeostasis, which is known to cause various diseases, including cancer. Maintaining precise control over the homeostasis of ISCs is crucial to preserve the intestinal epithelium's integrity during homeostasis or stressed conditions. Recent research has indicated that nutrients and metabolic pathways can extensively modulate the fate of ISCs. This review will explore recent findings concerning the influence of various nutrients, including lipids, carbohydrates, and vitamin D, on the delicate balance between ISC proliferation and differentiation.
    DOI:  https://doi.org/10.1111/cpr.13602
  17. bioRxiv. 2024 Feb 07. pii: 2024.02.06.579216. [Epub ahead of print]
    mTORC1 activity oscillates throughout the cell cycle promoting mitotic entry and differentially influencing autophagy induction.
    Jay N Joshi, Ariel D Lerner, Frank Scallo, Alexandra N Grumet, Paul Matteson, James H Millonig, Alexander J Valvezan.
      Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that stimulates anabolic cell growth while suppressing catabolic processes such as autophagy. mTORC1 is active in most, if not all, proliferating eukaryotic cells. However, it remains unclear whether and how mTORC1 activity changes from one cell cycle phase to another. Here we tracked mTORC1 activity through the complete cell cycle and uncover oscillations in its activity. We find that mTORC1 activity peaks in S and G2, and is lowest in mitosis and G1. We further demonstrate that multiple mechanisms are involved in controlling this oscillation. The interphase oscillation is mediated through the TSC complex, an upstream negative regulator of mTORC1, but is independent of major known regulatory inputs to the TSC complex, including Akt, Mek/Erk, and CDK4/6 signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex, and instead involves CDK1-dependent control of the subcellular localization of mTORC1 itself. Functionally, we find that in addition to its well-established role in promoting progression through G1, mTORC1 also promotes progression through S and G2, and is important for satisfying the Wee1- and Chk1-dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific functional consequences in proliferating cells.
    DOI:  https://doi.org/10.1101/2024.02.06.579216
  18. Front Immunol. 2024 ;15 1321321
    Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.
    Vadym Sulimenko, Vladimíra Sládková, Tetyana Sulimenko, Eduarda Dráberová, Věra Vosecká, Lubica Dráberová, Omar Skalli, Pavel Dráber.
      Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.
    Keywords:  G protein-coupled receptor kinase-interacting protein 2 (GIT2); centrosome; mast cells; microtubule nucleation; protein kinase C (PKC)
    DOI:  https://doi.org/10.3389/fimmu.2024.1321321
  19. Life Sci Alliance. 2024 May;pii: e202302300. [Epub ahead of print]7(5):
    Ageing-dependent thiol oxidation reveals early oxidation of proteins with core proteostasis functions.
    Katarzyna Jonak, Ida Suppanz, Julian Bender, Agnieszka Chacinska, Bettina Warscheid, Ulrike Topf.
      Oxidative post-translational modifications of protein thiols are well recognized as a readily occurring alteration of proteins, which can modify their function and thus control cellular processes. The development of techniques enabling the site-specific assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early-oxidation targets in key biological processes governing the de novo production of proteins, protein folding, and degradation, and indicate a hierarchy of cellular responses affected by a reversible redox modification. Comparison with existing datasets in yeast, nematode, fruit fly, and mouse reveals the evolutionary conservation of these oxidation targets. To facilitate accessibility, we integrated the cross-species comparison into the newly developed OxiAge Database.
    DOI:  https://doi.org/10.26508/lsa.202302300
  20. Chem Soc Rev. 2024 Feb 19.
    Targeted protein degradation directly engaging lysosomes or proteasomes.
    Jiseong Kim, Insuk Byun, Do Young Kim, Hyunhi Joh, Hak Joong Kim, Min Jae Lee.
      Targeted protein degradation (TPD) has been established as a viable alternative to attenuate the function of a specific protein of interest in both biological and clinical contexts. The unique TPD mode-of-action has allowed previously undruggable proteins to become feasible targets, expanding the landscape of "druggable" properties and "privileged" target proteins. As TPD continues to evolve, a range of innovative strategies, which do not depend on recruiting E3 ubiquitin ligases as in proteolysis-targeting chimeras (PROTACs), have emerged. Here, we present an overview of direct lysosome- and proteasome-engaging modalities and discuss their perspectives, advantages, and limitations. We outline the chemical composition, biochemical activity, and pharmaceutical characteristics of each degrader. These alternative TPD approaches not only complement the first generation of PROTACs for intracellular protein degradation but also offer unique strategies for targeting pathologic proteins located on the cell membrane and in the extracellular space.
    DOI:  https://doi.org/10.1039/d3cs00344b
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