bims-proteo Biomed News
on Proteostasis
Issue of 2022‒08‒28
twenty-six papers selected by
Eric Chevet
INSERM
  1. A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.
  2. Ultrastructure of COPII vesicle formation in yeast characterised by correlative light and electron microscopy.
  3. ER, Mitochondria, and ISR Regulation by mt-HSP70 and ATF5 upon Procollagen Misfolding in Osteoblasts.
  4. Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2.
  5. Quantitative Assessment of Arsenite-Induced Perturbation of Ubiquitinated Proteome.
  6. LEAP: a novel LC3C-dependent pathway connects autophagy, endocytic trafficking and signaling.
  7. In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair.
  8. A Nedd4 E3 Ubiquitin ligase pathway inhibits Robo1 repulsion and promotes commissural axon guidance across the midline.
  9. UDP-glucose, cereblon-dependent proinsulin degrader.
  10. HSF1, Aging, and Neurodegeneration.
  11. Hyperactivation of the proteasome in Caenorhabditis elegans protects against proteotoxic stress and extends lifespan.
  12. Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.
  13. Discovery of XL01126: A Potent, Fast, Cooperative, Selective, Orally Bioavailable, and Blood-Brain Barrier Penetrant PROTAC Degrader of Leucine-Rich Repeat Kinase 2.
  14. Proteasomal degradation of wild-type proinsulin in pancreatic beta cells.
  15. Restoring TRAILR2/DR5-Mediated Activation of Apoptosis upon Endoplasmic Reticulum Stress as a Therapeutic Strategy in Cancer.
  16. Autophagy regulates rRNA synthesis.
  17. Identification of chemicals breaking the USP8 interaction with its endocytic substrate CHMP1B.
  18. Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons.
  19. A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes.
  20. The amino acid sensor GCN2 controls red blood cell clearance and iron metabolism through regulation of liver macrophages.
  21. Increasing cell size remodels the proteome and promotes senescence.
  22. PROTAC degraders as chemical probes for studying target biology and target validation.
  23. Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia.
  24. Emerging Roles for G-Quadruplexes in Proteostasis.
  25. Identification of a drug-response gene in multiple myeloma through longitudinal single-cell transcriptome sequencing.
  26. Lysosomal exocytosis releases pathogenic α-synuclein species from neurons in synucleinopathy models.
  1. Mol Cell. 2022 Aug 20. pii: S1097-2765(22)00756-0. [Epub ahead of print]
    A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.
    Vishnu Balaji, Leonie Müller, Robin Lorenz, Éva Kevei, William H Zhang, Ulises Santiago, Jan Gebauer, Ernesto Llamas, David Vilchez, Carlos J Camacho, Wojciech Pokrzywa, Thorsten Hoppe.
      The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.
    Keywords:  C. elegans; CHIP; DAF-2; E3 ligase; aging; chaperones; insulin signaling; longevity; proteostasis; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2022.08.003
  2. Mol Biol Cell. 2022 Aug 24. mbcE22030103
    Ultrastructure of COPII vesicle formation in yeast characterised by correlative light and electron microscopy.
    Alejandro Melero, Jerome Boulanger, Wanda Kukulski, Elizabeth A Miller.
      Traffic of proteins out of the endoplasmic reticulum (ER) is driven by the COPII coat, a layered protein scaffold that mediates the capture of cargo proteins and the remodelling of the ER membrane into spherical vesicular carriers. Although the components of this machinery have been genetically defined, and the mechanisms of coat assembly extensively explored in vitro, understanding the physical mechanisms of membrane remodelling in cells remains a challenge. Here we use correlative light and electron microscopy (CLEM) to visualize the nanoscale ultrastructure of membrane remodelling at ER exit sites (ERES) in yeast cells. Using various COPII mutants, we have determined the broad contribution that each layer of the coat makes in membrane remodelling. Our data suggest that inner coat components define the radius of curvature whereas outer coat components facilitate membrane fission. The organization of the coat in conjunction with membrane biophysical properties determine the ultrastructure of vesicles and thus the efficiency of protein transport. [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-03-0103
  3. Adv Sci (Weinh). 2022 Aug 21. e2201273
    ER, Mitochondria, and ISR Regulation by mt-HSP70 and ATF5 upon Procollagen Misfolding in Osteoblasts.
    Laura Gorrell, Elena Makareeva, Shakib Omari, Satoru Otsuru, Sergey Leikin.
      Cellular response to protein misfolding underlies multiple diseases. Collagens are the most abundant vertebrate proteins, yet little is known about cellular response to misfolding of their procollagen precursors. Osteoblasts (OBs)-the cells that make bone-produce so much procollagen that it accounts for up to 40% of mRNAs in the cell, which is why bone bears the brunt of mutations causing procollagen misfolding in osteogenesis imperfecta (OI). The present study of a G610C mouse model of OI by multiple transcriptomic techniques provides first solid clues to how OBs respond to misfolded procollagen accumulation in the endoplasmic reticulum (ER) and how this response affects OB function. Surprisingly, misfolded procollagen escapes the quality control in the ER lumen and indirectly triggers the integrated stress response (ISR) through other cell compartments. In G610C OBs, the ISR is regulated by mitochondrial HSP70 (mt-HSP70) and ATF5 instead of their BIP and ATF4 paralogues, which normally activate and regulate ISR to secretory protein misfolding in the ER. The involvement of mt-HSP70 and ATF5 together with other transcriptomic findings suggest that mitochondria might initiate the ISR upon disruption of ER-mitochondria connections or might respond to the ISR activated by a yet unknown sensor.
    Keywords:  ATF5; HSPA9/mt-Hsp70/GRP75; cell stress; collagen; osteoblast; osteogenesis imperfecta
    DOI:  https://doi.org/10.1002/advs.202201273
  4. EMBO J. 2022 Aug 22. e110777
    Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2.
    Norbert Volkmar, Christian M Gawden-Bone, James C Williamson, Jonathon Nixon-Abell, James A West, Peter H St George-Hyslop, Arthur Kaser, Paul J Lehner.
      The regulation of membrane lipid composition is critical for cellular homeostasis. Cells are particularly sensitive to phospholipid saturation, with increased saturation causing membrane rigidification and lipotoxicity. How mammalian cells sense membrane lipid composition and reverse fatty acid (FA)-induced membrane rigidification is poorly understood. Here we systematically identify proteins that differ between mammalian cells fed saturated versus unsaturated FAs. The most differentially expressed proteins were two ER-resident polytopic membrane proteins: the E3 ubiquitin ligase RNF145 and the lipid hydrolase ADIPOR2. In unsaturated lipid membranes, RNF145 is stable, promoting its lipid-sensitive interaction, ubiquitination and degradation of ADIPOR2. When membranes become enriched in saturated FAs, RNF145 is rapidly auto-ubiquitinated and degraded, stabilising ADIPOR2, whose hydrolase activity restores lipid homeostasis and prevents lipotoxicity. We therefore identify RNF145 as a FA-responsive ubiquitin ligase which, together with ADIPOR2, defines an autoregulatory pathway that controls cellular membrane lipid homeostasis and prevents acute lipotoxic stress.
    Keywords:  RNF145; adiponectin receptor 2; lipid homoestasis; membrane rigidity; palmitic acid
    DOI:  https://doi.org/10.15252/embj.2022110777
  5. Chem Res Toxicol. 2022 Aug 22.
    Quantitative Assessment of Arsenite-Induced Perturbation of Ubiquitinated Proteome.
    Ji Jiang, Yinsheng Wang.
      Arsenic contamination in food and groundwater constitutes a public health concern for more than 200 million people worldwide. Individuals chronically exposed to arsenic through drinking and ingestion exhibit a higher risk of developing cancers and cardiovascular diseases. Nevertheless, the underlying mechanisms of arsenic toxicity are not fully understood. Arsenite is known to bind to and deactivate RING finger E3 ubiquitin ligases; thus, we reason that a systematic interrogation about how arsenite exposure modulates global protein ubiquitination may reveal novel molecular targets for arsenic toxicity. By employing liquid chromatography-tandem mass spectrometry, in combination with stable isotope labeling by amino acids in cell culture (SILAC) and immunoprecipitation of di-glycine-conjugated lysine-containing tryptic peptides, we assessed the alterations in protein ubiquitination in GM00637 human skin fibroblast cells upon arsenite exposure at the entire proteome level. We observed that arsenite exposure led to altered ubiquitination of many proteins, where the alterations in a large majority of ubiquitination events are negatively correlated with changes in expression of the corresponding proteins, suggesting their modulation by the ubiquitin-proteasomal pathway. Moreover, we observed that arsenite exposure confers diminished ubiquitination of a rate-limiting enzyme in cholesterol biosynthesis, HMGCR, at Lys248. We also revealed that TRC8 is the major E3 ubiquitin ligase for HMGCR ubiquitination in HEK293T cells, and the arsenite-induced diminution of HMGCR ubiquitination is abrogated upon genetic depletion of TRC8. In summary, we systematically characterized arsenite-induced perturbations in a ubiquitinated proteome in human cells and found that the arsenite-elicited attenuation of HMGCR ubiquitination in HEK293T cells involves TRC8.
    DOI:  https://doi.org/10.1021/acs.chemrestox.2c00197
  6. Autophagy. 2022 Aug 26.
    LEAP: a novel LC3C-dependent pathway connects autophagy, endocytic trafficking and signaling.
    Paula P Coelho, Morag Park.
      Macroautophagy/autophagy acts to promote homeostasis and is increasingly understood to selectively target cargo for degradation. The LC3-family of proteins mediate diverse yet distinct cargo recruitment to phagophores. However, what underlies specificity for cargo engagement among LC3 proteins is poorly understood. Using an unbiased protein interaction screen of LC3B and LC3C we uncover a novel LC3C-endocytic-associated-pathway (LEAP) that recruits selective plasma membrane (PM) cargo to phagophores. We show LC3C but not LC3B localizes to peripheral endosomes and engages proteins that traffic between the PM, endosomes and autophagosomes. We establish that endocytic LC3C binds cargo internalized from the PM, including MET receptor tyrosine kinase and TFRC (transferrin receptor), and targets them towards autophagic degradation. These findings identify LEAP as an unexpected LC3C-dependent pathway, providing new understanding of selective coupling of PM signaling and autophagic degradation with important implications in cancer and other disease states.
    Keywords:  Atg8-orthologs; LC3C; MET-RTK; autophagy; endocytic trafficking; selective cargo recruitment; signalophagy
    DOI:  https://doi.org/10.1080/15548627.2022.2117973
  7. Proc Natl Acad Sci U S A. 2022 Aug 30. 119(35): e2205590119
    In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair.
    Sankalp Shukla, Kevin P Larsen, Chenxi Ou, Kevin Rose, James H Hurley.
      The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.
    Keywords:  in vitro reconstitution; membrane biology; membrane remodeling; membrane repair; neurodegeneration
    DOI:  https://doi.org/10.1073/pnas.2205590119
  8. J Neurosci. 2022 Aug 22. pii: JN-RM-2491-21. [Epub ahead of print]
    A Nedd4 E3 Ubiquitin ligase pathway inhibits Robo1 repulsion and promotes commissural axon guidance across the midline.
    Madhavi Gorla, Karina Chaudhari, Maya Hale, Chloe Potter, Greg J Bashaw.
      Commissural axons initially respond to attractive signals at the midline, but once they cross, they become sensitive to repulsive cues. In insects and mammals, negative regulation of the surface expression of Roundabout (Robo) receptors prevents premature response to Slit. We previously identified two mammalian Nedd4 interacting proteins, Ndfip1 and Ndfip2, that act analogously to Drosophila Commissureless (Comm) to recruit mammalian Robo1 to late endosomes. However, whether Nedd4 E3 ubiquitin ligases are required for Ndfip-mediated Robo1 regulation and midline axon crossing in vivo is not known. Here we show using in vitro biochemical techniques and genetic analysis using embryonic mice of either sex that Nedd4-1 and Nedd4-2 are specifically required for Robo1 regulation and spinal commissural axon guidance. Biochemical data indicate that Robo1, Ndfip and Nedd4 form a ternary protein complex that depends on the presence of Ndfip, and these interactions are required for Robo1 endosomal sorting, ubiquitylation and degradation. Nedd4-1 and Nedd4-2 are expressed in commissural neurons in the developing spinal cord, and conditional deletion of Nedd4-1 or Nedd4-2 results in dose-dependent defects in midline crossing. We propose that Nedd4 E3 Ubiquitin ligases and their adaptor proteins Ndfip1 and Ndfip2 constitute a vital intracellular trafficking pathway required to down-regulate Robo1 and promote midline crossing of commissural axons.Significance Statement:During the development of the nervous system, many neurons extend their axons across the midline to establish circuits that are important for sensory, motor and cognitive functions. In order to cross the midline, axon responses to midline-derived cues must be precisely regulated. Here we characterize an important intracellular trafficking pathway that regulates the membrane expression of the conserved Roundabout axon guidance receptor- the receptor for the midline repellant Slit. We show that Nedd4 E3 Ubiquitin ligases and their Ndfip adapter proteins inhibit premature responses to Slit by promoting Robo degradation in pre-crossing commissural neurons in the developing spinal cord.
    DOI:  https://doi.org/10.1523/JNEUROSCI.2491-21.2022
  9. Sci Rep. 2022 Aug 26. 12(1): 14568
    UDP-glucose, cereblon-dependent proinsulin degrader.
    Jaeyong Cho, Atsushi Miyagawa, Kazuki Yamaguchi, Wakana Abe, Yoji Tsugawa, Hatsuo Yamamura, Takeshi Imai.
      Insulin secretion is regulated in multiple steps, and one of the main steps is in the endoplasmic reticulum (ER). Here, we show that UDP-glucose induces proinsulin ubiquitination by cereblon, and uridine binds and competes for proinsulin degradation and behaves as sustainable insulin secretagogue. Using insulin mutagenesis of neonatal diabetes variant-C43G and maturity-onset diabetes of the young 10 (MODY10) variant-R46Q, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) protects cereblon-dependent proinsulin ubiquitination in the ER. Cereblon is a ligand-inducible E3 ubiquitin ligase, and we found that UDP-glucose is the first identified endogenous proinsulin protein degrader. Uridine-containing compounds, such as uridine, UMP, UTP, and UDP-galactose, inhibit cereblon-dependent proinsulin degradation and stimulate insulin secretion from 3 to 24 h after administration in β-cell lines as well as mice. This late and long-term insulin secretion stimulation is designated a day sustainable insulin secretion stimulation. Uridine-containing compounds are designated as proinsulin degradation regulators.
    DOI:  https://doi.org/10.1038/s41598-022-18902-5
  10. Adv Exp Med Biol. 2022 Aug 23.
    HSF1, Aging, and Neurodegeneration.
    Alice Y Liu, Conceição A Minetti, David P Remeta, Kenneth J Breslauer, Kuang Yu Chen.
      Heat shock factor 1 (HSF1) is a master transcription regulator that mediates the induction of heat shock protein chaperones for quality control (QC) of the proteome and maintenance of proteostasis as a protective mechanism in response to stress. Research in this particular area has accelerated dramatically over the past three decades following successful isolation, cloning, and characterization of HSF1. The intricate multi-protein complexes and transcriptional activation orchestrated by HSF1 are fundamental processes within the cellular QC machinery. Our primary focus is on the regulation and function of HSF1 in aging and neurodegenerative diseases (ND) which represent physiological and pathological states of dysfunction in protein QC. This chapter presents an overview of HSF1 structural, functional, and energetic properties in healthy cells while addressing the deterioration of HSF1 function viz-à-viz age-dependent and neuron-specific vulnerability to ND. We discuss the structural domains of HSF1 with emphasis on the intrinsically disordered regions and note that disease proteins associated with ND are often structurally disordered and exquisitely sensitive to changes in cellular environment as may occur during aging. We propose a hypothesis that age-dependent changes of the intrinsically disordered proteome likely hold answers to understand many of the functional, structural, and organizational changes of proteins and signaling pathways in aging - dysfunction of HSF1 and accumulation of disease protein aggregates in ND included.Structured AbstractsIntroduction: Heat shock factor 1 (HSF1) is a master transcription regulator that mediates the induction of heat shock protein chaperones for quality control (QC) of the proteome as a cyto-protective mechanism in response to stress. There is cumulative evidence of age-related deterioration of this QC mechanism that contributes to disease vulnerability.OBJECTIVES: Herein we discuss the regulation and function of HSF1 as they relate to the pathophysiological changes of protein quality control in aging and neurodegenerative diseases (ND).
    METHODS: We present an overview of HSF1 structural, functional, and energetic properties in healthy cells while addressing the deterioration of HSF1 function vis-à-vis age-dependent and neuron-specific vulnerability to neurodegenerative diseases.
    RESULTS: We examine the impact of intrinsically disordered regions on the function of HSF1 and note that proteins associated with neurodegeneration are natively unstructured and exquisitely sensitive to changes in cellular environment as may occur during aging.
    CONCLUSIONS: We put forth a hypothesis that age-dependent changes of the intrinsically disordered proteome hold answers to understanding many of the functional, structural, and organizational changes of proteins - dysfunction of HSF1 in aging and appearance of disease protein aggregates in neurodegenerative diseases included.
    Keywords:  Aging; HSF1, Heat shock factor 1; HSP, heat shock protein family; Hsp, Specific heat shock protein; Intrinsically disordered proteome; Neurodegeneration; Protein homeostasis
    DOI:  https://doi.org/10.1007/5584_2022_733
  11. J Biol Chem. 2022 Aug 22. pii: S0021-9258(22)00858-4. [Epub ahead of print] 102415
    Hyperactivation of the proteasome in Caenorhabditis elegans protects against proteotoxic stress and extends lifespan.
    Raymond T Anderson, Thomas A Bradley, David M Smith.
      Virtually all age-related neurodegenerative diseases (NDs) can be characterized by the accumulation of proteins inside and outside the cell that are thought to significantly contribute to disease pathogenesis. One of the cell's primary systems for the degradation of misfolded/damaged proteins is the Ubiquitin Proteasome System (UPS), and its impairment is implicated in essentially all NDs. Thus, upregulating this system to combat NDs has garnered a great deal of interest in recent years. Various animal models have focused on stimulating 26S activity and increasing 20S proteasome levels, but thus far, none have targeted intrinsic activation of the 20S proteasome itself. Therefore, we constructed an animal model that endogenously expresses a hyperactive, open-gate proteasome in Caenorhabditis elegans (C. elegans). The gate-destabilizing mutation that we introduced into the nematode germline yielded a viable nematode population with enhanced proteasomal activity, including peptide, unstructured protein, and ubiquitin-dependent degradation activities. We determined these nematodes showed a significantly increased lifespan and substantial resistance to oxidative and proteotoxic stress but a significant decrease in fecundity. Our results show that introducing a constitutively active proteasome into a multicellular organism is feasible and suggests targeting the proteasome gating mechanism as a valid approach for future age-related disease research efforts in mammals.
    Keywords:  Aging; Caenorhabditis elegans (C. elegans); Enzyme kinetics; Oxidative stress; Proteasome; Protein degradation; Proteotoxic stress; Toxicity; Ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2022.102415
  12. Science. 2022 Aug 25. eabg6621
    Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.
    Hijai R Shin, Y Rose Citron, Lei Wang, Laura Tribouillard, Claire S Goul, Robin Stipp, Yusuke Sugasawa, Aakriti Jain, Nolwenn Samson, Chun-Yan Lim, Oliver B Davis, David Castaneda-Carpio, Mingxing Qian, Daniel K Nomura, Rushika M Perera, Eunyong Park, Douglas F Covey, Mathieu Laplante, Alex S Evers, Roberto Zoncu.
      Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified LYsosomal CHOlesterol Signaling (LYCHOS, previously annotated as G-protein coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1). Cholesterol bound to the N-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a GTPase-activating protein for the Rag guanosine triphosphatases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing, and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.
    DOI:  https://doi.org/10.1126/science.abg6621
  13. J Am Chem Soc. 2022 Aug 25.
    Discovery of XL01126: A Potent, Fast, Cooperative, Selective, Orally Bioavailable, and Blood-Brain Barrier Penetrant PROTAC Degrader of Leucine-Rich Repeat Kinase 2.
    Xingui Liu, Alexia F Kalogeropulou, Sofia Domingos, Nikolai Makukhin, Raja S Nirujogi, Francois Singh, Natalia Shpiro, Anton Saalfrank, Esther Sammler, Ian G Ganley, Rui Moreira, Dario R Alessi, Alessio Ciulli.
      Leucine-rich repeat kinase 2 (LRRK2) is one of the most promising targets for Parkinson's disease. LRRK2-targeting strategies have primarily focused on type 1 kinase inhibitors, which, however, have limitations as the inhibited protein can interfere with natural mechanisms, which could lead to undesirable side effects. Herein, we report the development of LRRK2 proteolysis targeting chimeras (PROTACs), culminating in the discovery of degrader XL01126, as an alternative LRRK2-targeting strategy. Initial designs and screens of PROTACs based on ligands for E3 ligases von Hippel-Lindau (VHL), Cereblon (CRBN), and cellular inhibitor of apoptosis (cIAP) identified the best degraders containing thioether-conjugated VHL ligand VH101. A second round of medicinal chemistry exploration led to qualifying XL01126 as a fast and potent degrader of LRRK2 in multiple cell lines, with DC50 values within 15-72 nM, Dmax values ranging from 82 to 90%, and degradation half-lives spanning from 0.6 to 2.4 h. XL01126 exhibits high cell permeability and forms a positively cooperative ternary complex with VHL and LRRK2 (α = 5.7), which compensates for a substantial loss of binary binding affinities to VHL and LRRK2, underscoring its strong degradation performance in cells. Remarkably, XL01126 is orally bioavailable (F = 15%) and can penetrate the blood-brain barrier after either oral or parenteral dosing in mice. Taken together, these experiments qualify XL01126 as a suitable degrader probe to study the noncatalytic and scaffolding functions of LRRK2 in vitro and in vivo and offer an attractive starting point for future drug development.
    DOI:  https://doi.org/10.1021/jacs.2c05499
  14. J Biol Chem. 2022 Aug 18. pii: S0021-9258(22)00849-3. [Epub ahead of print] 102406
    Proteasomal degradation of wild-type proinsulin in pancreatic beta cells.
    Xiaoxi Xu, Anoop Arunagiri, Leena Haataja, Maroof Alam, Shuhui Ji, Ling Qi, Billy Tsai, Ming Liu, Peter Arvan.
      Preproinsulin entry into the endoplasmic reticulum yields proinsulin, and its subsequent delivery to the distal secretory pathway leads to processing, storage, and secretion of mature insulin. Multiple groups have reported that treatment of pancreatic beta cell lines, rodent pancreatic islets, or human islets with proteasome inhibitors leads to diminished proinsulin and insulin protein levels, diminished glucose-stimulated insulin secretion, and changes in beta cell gene expression that ultimately lead to beta cell death. However, these studies have mostly examined treatment times far beyond that needed to achieve acute proteasomal inhibition. Here, we report that although proteasomal inhibition immediately downregulates new proinsulin biosynthesis, it nevertheless acutely increases beta cell proinsulin levels in pancreatic beta cell lines, rodent pancreatic islets, and human islets, indicating rescue of a pool of recently-synthesized wildtype INS gene product that would otherwise be routed to proteasomal disposal. Our pharmacological evidence suggests that this disposal most likely reflects ongoing ER-associated protein degradation. However, we found that within 60 minutes after proteasomal inhibition, intracellular proinsulin levels begin to fall in conjunction with increased phosphorylation of eIF2α, which can be inhibited by blocking the GCN2 kinase. Together, these data demonstrate that a meaningful sub-fraction of newly-synthesized INS gene product undergoes rapid proteasomal disposal. We propose that free amino acids derived from proteasomal proteolysis may potentially participate in suppressing GCN2 kinase activity to maintain ongoing proinsulin biosynthesis.
    Keywords:  ERAD; cell stress; endoplasmic reticulum; insulin production; secretory pathway
    DOI:  https://doi.org/10.1016/j.jbc.2022.102406
  15. Int J Mol Sci. 2022 Aug 12. pii: 8987. [Epub ahead of print]23(16):
    Restoring TRAILR2/DR5-Mediated Activation of Apoptosis upon Endoplasmic Reticulum Stress as a Therapeutic Strategy in Cancer.
    Rocío Mora-Molina, Abelardo López-Rivas.
      The uncontrolled proliferation of malignant cells in growing tumors results in the generation of different stressors in the tumor microenvironment, such as nutrient shortage, hypoxia and acidosis, among others, that disrupt endoplasmic reticulum (ER) homeostasis and may lead to ER stress. As a response to ER stress, both normal and tumor cells launch a set of signaling pathways known as the unfolded protein response (UPR) to restore ER proteostasis and maintain cell viability and function. However, under sustained ER stress, an apoptotic cell death process can be induced and this has been the subject of different review articles, although the role of the TRAIL-R2/DR5-activated extrinsic pathway of apoptosis has not yet been thoroughly summarized. In this Review, we provide an updated overview of the molecular mechanisms regulating cell fate decisions in tumor cells undergoing ER stress and discuss the role of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5) in the final outcome of UPR signaling. Particularly, we focus on the mechanisms controlling cellular FLICE-like inhibitory protein (FLIP) levels in tumor cells undergoing ER stress, which may represent a potential target for therapeutic intervention in cancer.
    Keywords:  FLIP; TRAILR2/DR5; apoptosis; cancer; endoplasmic reticulum stress; extrinsic pathway; tumor microenvironment; unfolded protein response
    DOI:  https://doi.org/10.3390/ijms23168987
  16. Nucleus. 2022 Dec;13(1): 203-207
    Autophagy regulates rRNA synthesis.
    Yinfeng Xu, Wei Wan.
      Autophagy has emerged as a key regulator of cell metabolism. Recently, we have demonstrated that autophagy is involved in RNA metabolism by regulating ribosomal RNA (rRNA) synthesis. We found that autophagy-deficient cells display much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling, which facilitates the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoter regions and leads to the activation of rDNA transcription. Finally, we showed that SQSTM1 accumulation is responsible for the increase in protein synthesis, cell growth and cell proliferation in autophagy-deficient cells. Taken together, our findings reveal a regulatory role of autophagy and autophagy receptor SQSTM1 in rRNA synthesis and may provide novel mechanisms for the hyperactivated rDNA transcription in autophagy-related human diseases.Abbreviations: 5-FUrd: 5-fluorouridine; LAP: MAP1LC3/LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A: RNA polymerase I subunit A; rDNA: ribosomal DNA; RRN3: RRN3 homolog, RNA polymerase I transcription factor; rRNA: ribosomal RNA; SQSTM1/p62: sequestosome 1; TP53INP2: tumor protein p53 inducible nuclear protein 2; UBTF: upstream binding transcription factor.
    Keywords:  Autophagy; MTORC1; SQSTM1/p62; rDNA; rRNA
    DOI:  https://doi.org/10.1080/19491034.2022.2114661
  17. SLAS Discov. 2022 Aug 19. pii: S2472-5552(22)13687-7. [Epub ahead of print]
    Identification of chemicals breaking the USP8 interaction with its endocytic substrate CHMP1B.
    Journet Agnès, Barette Caroline, Aubry Laurence, Soleilhac Emmanuelle, Fauvarque Marie-Odile.
      The ubiquitin-specific protease USP8 plays a major role in controlling the stability and intracellular trafficking of numerous cell surface proteins among which the EGF receptor that regulates cell growth and proliferation in many physio-pathological processes. The function of USP8 at the endocytic pathway level partly relies on binding to and deubiquitination of the Endosomal Sorting Complex Required for Transport (ESCRT) protein CHMP1B. In the aim of finding chemical inhibitors of the USP8::CHMP1B interaction, we performed a high-throughput screening campaign using an HTRF® assay to monitor the interaction directly in lysates of cells co-expressing both partners. The assay was carried out in an automated format to screen the academic Fr-PPIChem library (Bosc N et al., 2020), which includes 10,314 compounds dedicated to the targeting of protein-protein interactions (PPIs). Eleven confirmed hits inhibited the USP8::CHMP1B interaction within a range of 30% to 70% inhibition at 50 µM, while they were inactive on a set of other PPI interfaces demonstrating the feasibility of specifically disrupting this particular interface. In parallel, we adapted this HTRF® assay to compare the USP8 interacting capacity of CHMP1B variants. As anticipated from earlier studies, a deletion of the MIM (Microtubule Interacting and Trafficking domain Interacting Motif) domain or mutation of two conserved leucine residues, L192 and L195, in this domain respectively abolished or strongly impeded the USP8::CHMP1B interaction. By contrast, a CHMP1B mutant that displays a highly decreased ubiquitination level following mutation of four lysine residues in arginine interacted at a similar level as the wild-type form with USP8. Therefore, conserved leucine residues within the MIT domain rather than its ubiquitinated status triggers CHMP1B substrate recognition by USP8.
    Keywords:  Drug discovery; ESCRT; Endocytosis; High-throughput screening; MIT domain; Protein-protein interaction
    DOI:  https://doi.org/10.1016/j.slasd.2022.08.003
  18. EMBO J. 2022 Aug 25. e110486
    Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons.
    Sumiko Kiryu-Seo, Reika Matsushita, Yoshitaka Tashiro, Takeshi Yoshimura, Yohei Iguchi, Masahisa Katsuno, Ryosuke Takahashi, Hiroshi Kiyama.
      The proteasome is essential for cellular responses to various physiological stressors. However, how proteasome function impacts the stress resilience of regenerative damaged motor neurons remains unclear. Here, we develop a unique mouse model using a regulatory element of the activating transcription factor (Atf3) gene to label mitochondria in a damage-induced manner while simultaneously genetically disrupting the proteasome. Using this model, we observed that in injury-induced proteasome-deficient mouse motor neurons, the increase of mitochondrial influx from soma into axons is inhibited because neurons fail to disassemble ankyrin G, an organizer of the axon initial segment (AIS), in a proteasome-dependent manner. Further, these motor neurons exhibit amyotrophic lateral sclerosis (ALS)-like degeneration despite having regenerative potential. Selectively vulnerable motor neurons in SOD1G93A ALS mice, which induce ATF3 in response to pathological damage, also fail to disrupt the AIS, limiting the number of axonal mitochondria at a pre-symptomatic stage. Thus, damage-induced proteasome-sensitive AIS disassembly could be a critical post-translational response for damaged motor neurons to temporarily transit to an immature state and meet energy demands for axon regeneration or preservation.
    Keywords:  axonal transport; nerve injury; neurodegenerative disease; organelle; stress response
    DOI:  https://doi.org/10.15252/embj.2021110486
  19. J Biol Chem. 2022 Aug 18. pii: S0021-9258(22)00837-7. [Epub ahead of print] 102394
    A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes.
    Francis K Fordjour, Chenxu Guo, Yiwei Ai, George G Daaboul, Stephen J Gould.
      Exosomes are small extracellular vesicles (EVs) of ∼30-150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes. We show that exosome cargo proteins bud from cells (i) in exosome-sized vesicles regardless of whether they are localized to plasma or endosome membranes, (ii) ∼5-fold more efficiently when localized to the plasma membrane, (iii) ∼5-fold less efficiently when targeted to the endosome membrane, (iv) by a stochastic process that leads to ∼100-fold differences in their abundance from one exosome to another, and (v) independently of small GTPase Rab27a, the ESCRT complex-associated protein Alix, or the cargo protein CD63. Taken together, our results demonstrate that cells use a shared, stochastic mechanism to bud exosome cargoes along the spectrum of plasma and endosome membranes, and far more efficiently from the plasma membrane than the endosome. Our observations also indicate that the pronounced variation in content between different exosome-sized vesicles is an inevitable consequence of a stochastic mechanism of small vesicle biogenesis, that the origin membrane of exosome-sized EVs simply cannot be determined, and that most of what we currently know about exosomes has likely come from studies of plasma membrane-derived vesicles.
    Keywords:  CD63; CD81; CD9; Protein budding; Rab27a; SPIR; endosome; extracellular vesicle; interferometric reflectance; interferometry; plasma membrane; tetraspanin
    DOI:  https://doi.org/10.1016/j.jbc.2022.102394
  20. Proc Natl Acad Sci U S A. 2022 Aug 30. 119(35): e2121251119
    The amino acid sensor GCN2 controls red blood cell clearance and iron metabolism through regulation of liver macrophages.
    Phoenix Toboz, Mehdi Amiri, Negar Tabatabaei, Catherine R Dufour, Seung Hyeon Kim, Carine Fillebeen, Charles E Ayemoba, Arkady Khoutorsky, Manfred Nairz, Lijian Shao, Kostandin V Pajcini, Ki-Wook Kim, Vincent Giguère, Regiana L Oliveira, Marco Constante, Manuela M Santos, Carlos R Morales, Kostas Pantopoulos, Nahum Sonenberg, Sandra Pinho, Soroush Tahmasebi.
      GCN2 (general control nonderepressible 2) is a serine/threonine-protein kinase that controls messenger RNA translation in response to amino acid availability and ribosome stalling. Here, we show that GCN2 controls erythrocyte clearance and iron recycling during stress. Our data highlight the importance of liver macrophages as the primary cell type mediating these effects. During different stress conditions, such as hemolysis, amino acid deficiency or hypoxia, GCN2 knockout (GCN2-/-) mice displayed resistance to anemia compared with wild-type (GCN2+/+) mice. GCN2-/- liver macrophages exhibited defective erythrophagocytosis and lysosome maturation. Molecular analysis of GCN2-/- cells demonstrated that the ATF4-NRF2 pathway is a critical downstream mediator of GCN2 in regulating red blood cell clearance and iron recycling.
    Keywords:  GCN2; RBC; hemolytic stress; mRNA translation; macrophages
    DOI:  https://doi.org/10.1073/pnas.2121251119
  21. Mol Cell. 2022 Aug 15. pii: S1097-2765(22)00713-4. [Epub ahead of print]
    Increasing cell size remodels the proteome and promotes senescence.
    Michael C Lanz, Evgeny Zatulovskiy, Matthew P Swaffer, Lichao Zhang, Ilayda Ilerten, Shuyuan Zhang, Dong Shin You, Georgi Marinov, Patrick McAlpine, Joshua E Elias, Jan M Skotheim.
      Cell size is tightly controlled in healthy tissues, but it is unclear how deviations in cell size affect cell physiology. To address this, we measured how the cell's proteome changes with increasing cell size. Size-dependent protein concentration changes are widespread and predicted by subcellular localization, size-dependent mRNA concentrations, and protein turnover. As proliferating cells grow larger, concentration changes typically associated with cellular senescence are increasingly pronounced, suggesting that large size may be a cause rather than just a consequence of cell senescence. Consistent with this hypothesis, larger cells are prone to replicative, DNA-damage-induced, and CDK4/6i-induced senescence. Size-dependent changes to the proteome, including those associated with senescence, are not observed when an increase in cell size is accompanied by an increase in ploidy. Together, our findings show how cell size could impact many aspects of cell physiology by remodeling the proteome and provide a rationale for cell size control and polyploidization.
    Keywords:  DNA damage; SA-beta-Gal; cell cycle; cell size; p16(INK4); palbociclib; polyploidy; proteomics; senescence; size-scaling
    DOI:  https://doi.org/10.1016/j.molcel.2022.07.017
  22. Chem Soc Rev. 2022 Aug 25.
    PROTAC degraders as chemical probes for studying target biology and target validation.
    Václav Němec, Martin P Schwalm, Susanne Müller, Stefan Knapp.
      Small molecule degraders such as PROTACs (PROteolysis TArgeting Chimeras) have emerged as new promising pharmacological modalities and the first PROTAC drug candidates are now studied clinically. The catalytic properties of PROTACs, acting as chemical degraders of a protein of interest (POI), represent an attractive new strategy for drug development. The development and characterization of PROTACs requires an array of additional assay systems that track the degradation pathway leading ultimately to degradation of the POI, identifying critical steps for PROTAC optimization. In addition to their exciting translational potential, PROTACs represent versatile chemical tools that considerably expanded our chemical biology toolbox and significantly enlarged the proteome that can be modulated by small molecules. Similar to conventional chemical probes, PROTACs used as chemical probes in target validation require comprehensive characterization. As a consequence, PROTAC-specific quality criteria should be defined by the chemical biology community. These criteria need to comprise additional or alternative parameters compared to those for conventional occupancy-driven chemical probes, such as the maximum level of target degradation (Dmax), confirmation of a proteasome dependent degradation mechanism and, importantly, also kinetic parameters of POI degradation. The kinetic aspects are particularly relevant for PROTACs that harbor covalent binding moieties. Here, we review recent progress in the development of assay systems for PROTAC characterization and suggest a set of criteria for PROTACs as high quality chemical probes.
    DOI:  https://doi.org/10.1039/d2cs00478j
  23. Leukemia. 2022 Aug 23.
    Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia.
    Sebastian E Koschade, Kevin Klann, Shabnam Shaid, Binje Vick, Jan A Stratmann, Marlyn Thölken, Laura M Meyer, The Duy Nguyen, Julia Campe, Laura M Moser, Susanna Hock, Fatima Baker, Christian T Meyer, Frank Wempe, Hubert Serve, Evelyn Ullrich, Irmela Jeremias, Christian Münch, Christian H Brandts.
      Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.
    DOI:  https://doi.org/10.1038/s41375-022-01678-y
  24. FEBS J. 2022 Aug 26.
    Emerging Roles for G-Quadruplexes in Proteostasis.
    Bryan B Guzman, Ahyun Son, Theodore J Litberg, Zijue Huang, Daniel Dominguez, Scott Horowitz.
      How nucleic acids interact with proteins, and how they affect protein folding, aggregation, and misfolding is a still-evolving area of research. Considerable effort is now focusing on a particular structure of RNA and DNA, G-quadruplexes, and their role in protein homeostasis and disease. In this State-of-the-Art Review, we track recent reports on how G-quadruplexes influence protein aggregation, proteolysis, phase separation, and protein misfolding diseases, and pose currently unanswered questions in the advance of this scientific field.
    Keywords:  ALS; Alzheimer; Fragile X; aggregation; chaperone; proteostasis; quadruplex
    DOI:  https://doi.org/10.1111/febs.16608
  25. iScience. 2022 Aug 19. 25(8): 104781
    Identification of a drug-response gene in multiple myeloma through longitudinal single-cell transcriptome sequencing.
    Toru Masuda, Shojiro Haji, Yasuhiro Nakashima, Mariko Tsuda, Daisaku Kimura, Akiko Takamatsu, Norifusa Iwahashi, Hironobu Umakoshi, Motoaki Shiratsuchi, Chie Kikutake, Mikita Suyama, Yasuyuki Ohkawa, Yoshihiro Ogawa.
      Despite recent therapeutic advances for multiple myeloma (MM), relapse is very common. Here, we conducted longitudinal single-cell transcriptome sequencing (scRNA-seq) of MM cells from a patient with relapsed MM, treated with multiple anti-myeloma drugs. We observed five subclusters of MM cells, which appeared and/or disappeared in response to the therapeutic pressure, and identified cluster 3 which emerged during lenalidomide treatment and disappeared after proteasome inhibitor (PI) treatment. Among the differentially expressed genes in cluster 3, we found a candidate drug-response gene; pellino E3 ubiquitin-protein ligase family member 2 (PELI2), which is responsible for PI-induced cell death in in vitro assay. Kaplan-Meier survival analysis of database revealed that higher expression of PELI2 is associated with a better prognosis. Our integrated strategy combining longitudinal scRNA-seq analysis, in vitro functional assay, and database analysis would facilitate the understanding of clonal dynamics of MM in response to anti-myeloma drugs and identification of drug-response genes.
    Keywords:  cancer; drugs; omics
    DOI:  https://doi.org/10.1016/j.isci.2022.104781
  26. Nat Commun. 2022 Aug 22. 13(1): 4918
    Lysosomal exocytosis releases pathogenic α-synuclein species from neurons in synucleinopathy models.
    Ying Xue Xie, Nima N Naseri, Jasmine Fels, Parinati Kharel, Yoonmi Na, Diane Lane, Jacqueline Burré, Manu Sharma.
      Considerable evidence supports the release of pathogenic aggregates of the neuronal protein α-Synuclein (αSyn) into the extracellular space. While this release is proposed to instigate the neuron-to-neuron transmission and spread of αSyn pathology in synucleinopathies including Parkinson's disease, the molecular-cellular mechanism(s) remain unclear. To study this, we generated a new mouse model to specifically immunoisolate neuronal lysosomes, and established a long-term culture model where αSyn aggregates are produced within neurons without the addition of exogenous fibrils. We show that neuronally generated pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons. We then find that neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis. The released aggregates are non-membrane enveloped and seeding-competent. Additionally, we find that this release is dependent on neuronal activity and cytosolic Ca2+. These results propose lysosomal exocytosis as a central mechanism for the release of aggregated and degradation-resistant proteins from neurons.
    DOI:  https://doi.org/10.1038/s41467-022-32625-1
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