bims-proteo Biomed News
on Proteostasis
Issue of 2022–12–25
25 papers selected by
Eric Chevet, INSERM
  1. Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation.
  2. Neurodegeneration Risk Factor, EIF2AK3 (PERK), Influences Tau Protein Aggregation.
  3. The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.
  4. The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER.
  5. Elucidation of ubiquitin-conjugating enzymes that interact with RBR-type ubiquitin ligases using a liquid-liquid phase separation-based method.
  6. The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER.
  7. Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA.
  8. Native size exclusion chromatography-based mass spectrometry (SEC-MS) reveals new components of the early Heat Shock Protein 90 inhibition response among limited global changes.
  9. The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress.
  10. Distribution and solvent exposure of Hsp70 chaperone binding sites across the E. coli proteome.
  11. The UBE2C/CDH1/DEPTOR axis is an oncogene-tumor suppressor cascade in lung cancer cells.
  12. Orchestration of selective autophagy by cargo receptors.
  13. Targeted protein degradation using the lysosomal pathway.
  14. Regulation of eIF4E guides a unique translational program to control erythroid maturation.
  15. Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia.
  16. Mitochondria and Endoplasmic Reticulum Stress in Retinal Organoids from Vision Loss Patients.
  17. Deregulation of HSF1-mediated endoplasmic reticulum unfolded protein response promotes cisplatin resistance in lung cancer cells.
  18. Recent Advances toward the Development of Hsp90 C-terminal Inhibitors.
  19. The unfolded protein response (UPR) pathway: the unsung hero in breast cancer management.
  20. Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children.
  21. Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays.
  22. Does the nuclear envelope retain its identity during mitosis?
  23. Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.
  24. A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity.
  25. Translational control by maternal Nanog promotes oogenesis and early embryonic development.
  1. Sci Adv. 2022 Dec 23. 8(51): eadd9520
    Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation.
    Erik Jonsson, Zaw Min Htet, Jared A M Bard, Ken C Dong, Andreas Martin.
      The 26S proteasome recognizes thousands of appropriate protein substrates in eukaryotic cells through attached ubiquitin chains and uses its adenosine triphosphatase (ATPase) motor for mechanical unfolding and translocation into a proteolytic chamber. Here, we used single-molecule Förster resonance energy transfer measurements to monitor the conformational dynamics of the proteasome, observe individual substrates during their progression toward degradation, and elucidate how these processes are regulated by ubiquitin chains. Rapid transitions between engagement- and processing-competent proteasome conformations control substrate access to the ATPase motor. Ubiquitin chain binding functions as an allosteric regulator to slow these transitions, stabilize the engagement-competent state, and aid substrate capture to accelerate degradation initiation. Upon substrate engagement, the proteasome remains in processing-competent states for translocation and unfolding, except for apparent motor slips when encountering stably folded domains. Our studies revealed how ubiquitin chains allosterically regulate degradation initiation, which ensures substrate selectivity in a crowded cellular environment.
    DOI:  https://doi.org/10.1126/sciadv.add9520
  2. J Biol Chem. 2022 Dec 20. pii: S0021-9258(22)01264-9. [Epub ahead of print] 102821
    Neurodegeneration Risk Factor, EIF2AK3 (PERK), Influences Tau Protein Aggregation.
    Goonho Park, Ke Xu, Leon Chea, Kyle Kim, Lance Safarta, Keon-Hyoung Song, Jian Wu, Soyoung Park, Hyejung Min, Nobuhiko Hiramatsu, Jaeseok Han, Jonathan H Lin.
      Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms - Unfolded Protein Response (UPR) and Integrated Stress Response (ISR). Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation while inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the ER luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.
    Keywords:  EIF2AK3; ER stress; Integrated Stress Response; Neurodegeneration; PERK; Tau aggregation; Tauopathy; Unfolded Protein Response; eIF2α phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2022.102821
  3. Nat Commun. 2022 Dec 21. 13(1): 7857
    The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.
    Ryosuke Ishimura, Afnan H El-Gowily, Daisuke Noshiro, Satoko Komatsu-Hirota, Yasuko Ono, Mayumi Shindo, Tomohisa Hatta, Manabu Abe, Takefumi Uemura, Hyeon-Cheol Lee-Okada, Tarek M Mohamed, Takehiko Yokomizo, Takashi Ueno, Kenji Sakimura, Tohru Natsume, Hiroyuki Sorimachi, Toshifumi Inada, Satoshi Waguri, Nobuo N Noda, Masaaki Komatsu.
      Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.
    DOI:  https://doi.org/10.1038/s41467-022-35501-0
  4. Cell Rep. 2022 Dec 20. pii: S2211-1247(22)01764-8. [Epub ahead of print]41(12): 111868
    The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER.
    Kou Motani, Noriko Saito-Tarashima, Kohei Nishino, Shunya Yamauchi, Noriaki Minakawa, Hidetaka Kosako.
      STING, an endoplasmic reticulum (ER)-resident receptor for cyclic di-nucleotides (CDNs), is essential for innate immune responses. Upon CDN binding, STING moves from the ER to the Golgi, where it activates downstream type-I interferon (IFN) signaling. General cargo proteins exit from the ER via concentration at ER exit sites. However, the mechanism of STING concentration is poorly understood. Here, we visualize the ER exit sites of STING by blocking its transport at low temperature or by live-cell imaging with the cell-permeable ligand bis-pivSATE-2'F-c-di-dAMP, which we have developed. After ligand binding, STING forms punctate foci at non-canonical ER exit sites. Unbiased proteomic screens and super-resolution microscopy show that the Golgi-resident protein ACBD3/GCP60 recognizes and concentrates ligand-bound STING at specialized ER-Golgi contact sites. Depletion of ACBD3 impairs STING ER-to-Golgi trafficking and type-I IFN responses. Our results identify the ACBD3-mediated non-canonical cargo concentration system that drives the ER exit of STING.
    Keywords:  ACBD3; APEX2; CP: Cell biology; CP: Immunology; ER exit site; ER-Golgi contact; ER-to-Golgi trafficking; STING; cyclic di-nucleotide; innate immune signaling; proximity proteomics; type-I interferon
    DOI:  https://doi.org/10.1016/j.celrep.2022.111868
  5. J Biol Chem. 2022 Dec 20. pii: S0021-9258(22)01265-0. [Epub ahead of print] 102822
    Elucidation of ubiquitin-conjugating enzymes that interact with RBR-type ubiquitin ligases using a liquid-liquid phase separation-based method.
    Ryota Hayashida, Reika Kikuchi, Kenichiro Imai, Waka Kojima, Tatsuya Yamada, Miho Iijima, Hiromi Sesaki, Keiji Tanaka, Noriyuki Matsuda, Koji Yamano.
      RBR-type ubiquitin ligases (E3s) such as Parkin receive ubiquitin from ubiquitin-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer ubiquitin to each RBR-type ligase are largely unknown due to insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a pro-inflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of ubiquitin-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.
    DOI:  https://doi.org/10.1016/j.jbc.2022.102822
  6. J Biol Chem. 2022 Dec 19. pii: S0021-9258(22)01256-X. [Epub ahead of print] 102813
    The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER.
    Saihas Suhda, Yasunori Yamamoto, Sindhu Wisesa, Risa Sada, Toshiaki Sakisaka.
      The reticular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions, and undergoes constant remodeling through formation and loss of the three-way junctions. TMCC3, an ER membrane protein localizing at three-way junctions, has been shown to positively regulate formation of the reticular ER network. However, elements that negatively regulate TMCC3 localization have not been characterized. In this study, we report that 14-3-3γ, a phospho-serine/phospho-threonine-binding protein involved in various signal transduction pathways, is a negative regulator of TMCC3. We demonstrate that overexpression of 14-3-3γ reduced localization of TMCC3 to three-way junctions, and decreased the number of three-way junctions. TMCC3 bound to 14-3-3γ through the N-terminus, and had deduced 14-3-3 binding motifs. Additionally, we determined that a TMCC3 mutant substituting alanine for serine to be phosphorylated in the binding motif reduced binding to 14-3-3γ. The TMCC3 mutant was more prone than wild-type TMCC3 to localize at three-way junctions in the cells overexpressing 14-3-3γ. Furthermore, the TMCC3 mutant rescued the ER sheet expansion caused by TMCC3 knockdown less than wild-type TMCC3. Taken together, these results indicate that 14-3-3γ binding negatively regulates localization of TMCC3 to the three-way junctions for the proper reticular ER network, implying that the negative regulation of TMCC3 by 14-3-3γ would underlie remodeling of the reticular network of the ER.
    Keywords:  14-3-3 protein; endoplasmic reticulum; membrane protein; protein phosphorylation; protein-protein interaction
    DOI:  https://doi.org/10.1016/j.jbc.2022.102813
  7. Mol Cell. 2022 Dec 13. pii: S1097-2765(22)01135-2. [Epub ahead of print]
    Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA.
    Gus D Warren, Tomoe Kitao, Tyler G Franklin, Justine V Nguyen, Paul P Geurink, Tomoko Kubori, Hiroki Nagai, Jonathan N Pruneda.
      The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.
    Keywords:  Legionella pneumophila; VCP/p97/Cdc48; bacterial effector; deubiquitinase; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2022.11.022
  8. Mol Cell Proteomics. 2022 Dec 19. pii: S1535-9476(22)00293-6. [Epub ahead of print] 100485
    Native size exclusion chromatography-based mass spectrometry (SEC-MS) reveals new components of the early Heat Shock Protein 90 inhibition response among limited global changes.
    Rahul S Samant, Silvia Batista, Mark Larance, Bugra Ozer, Christopher I Milton, Isabell Bludau, Estelle Wu, Laura Biggins, Simon Andrews, Alexia Hervieu, Harvey E Johnston, Bissan Al-Lazikhani, Angus I Lamond, Paul A Clarke, Paul Workman.
      The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 hours in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin, and the mitochondrial isocitrate dehydrogenase 3 (IDH3) complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.
    Keywords:  HSP90 inhibitor; heat shock protein; molecular chaperone; protein complexes; proteomics; tanespimycin
    DOI:  https://doi.org/10.1016/j.mcpro.2022.100485
  9. NPJ Precis Oncol. 2022 Dec 21. 6(1): 93
    The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress.
    Udayan Bhattacharya, Elangovan Thavathiru, Fiifi Neizer-Ashun, Chao Xu, Zoran Gatalica, Shailendra Kumar Dhar Dwivedi, Anindya Dey, Priyabrata Mukherjee, Resham Bhattacharya.
      The ubiquitin-specific peptidase 10 (USP10) plays a context-specific, pro or anti-tumorigenic role in different malignancies. However, the role of USP10 in pancreatic cancer remains unclear. Our protein and RNA level analysis from archived specimens and public databases show that USP10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and expression correlates with poor overall patient survival. Phenotypically, silencing USP10 decreased viability, clonal growth and invasive properties of pancreatic cancer cells. Mechanistically, silencing USP10 upregulated BiP and induced endoplasmic reticulum (ER) stress that led to an unfolded protein response (UPR) and upregulation of PERK, IRE1α. Decreased cell viability of USP10 silenced cells could be rescued by a chemical chaperone that promotes protein folding. Our studies suggest that USP10 by protecting pancreatic cancer cells from ER stress may support tumor progression.
    DOI:  https://doi.org/10.1038/s41698-022-00336-x
  10. Proteins. 2022 Dec 20.
    Distribution and solvent exposure of Hsp70 chaperone binding sites across the E. coli proteome.
    Xi Chen, Rachel B Hutchinson, Silvia Cavagnero.
      Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding-site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client-protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome-wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2,258 E. coli proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent-exposed than other nonpolar sites, in protein native states. Our findings show that the majority of E. coli proteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70-bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70-chaperone-binding traits have evolved to favor Hsp70-assisted protein folding devoid of aggregation.
    Keywords:  DnaK; Hsp70; binding site; chaperone; hydrophobicity; solubility; surface area
    DOI:  https://doi.org/10.1002/prot.26456
  11. J Clin Invest. 2022 Dec 22. pii: e162434. [Epub ahead of print]
    The UBE2C/CDH1/DEPTOR axis is an oncogene-tumor suppressor cascade in lung cancer cells.
    Shizhen Zhang, Xiahong You, Yawen Zheng, Yanwen Shen, Xiufang Xiong, Yi Sun.
      Ubiquitin-conjugating enzyme E2C (UBE2C) mediates the ubiquitylation chain formation via the K11 linkage. While previous in vitro studies showed that UBE2C plays a growth-promoting role in cancer cell lines, the underlying mechanism remains elusive. Still unknown is whether and how UBE2C plays a promoting role in vivo. Here we reported that UBE2C is indeed essential for growth and survival of lung cancer cells harboring Kras mutations, and UBE2C is required for KrasG12D-induced lung tumorigenesis, since Ube2c deletion significantly inhibits tumor formation and extends the life-span of mice. Mechanistically, KrasG12D induces expression of UBE2C, which couples with APC/CCDH1 E3 ligase to promote ubiquitylation and degradation of DEPTOR, leading to activation of the mTORC signals. Importantly, DEPTOR levels are fluctuated during cell cycle progression in a manner dependent of UBE2C and CDH1, indicating their physiological connection. Finally, Deptor deletion fully rescues the tumor inhibitory effect of Ube2c deletion in the KrasG12D lung tumor model, indicating a causal role of Deptor. Taken together, our study shows that the UBE2C/CDH1/DEPTOR axis forms an oncogene-tumor suppressor cascade that regulates cell cycle progression and autophagy and validates that UBE2C is an attractive target for lung cancer associated with Kras mutations.
    Keywords:  Cell Biology; Lung cancer; Oncology; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI162434
  12. Curr Biol. 2022 Dec 19. pii: S0960-9822(22)01758-4. [Epub ahead of print]32(24): R1357-R1371
    Orchestration of selective autophagy by cargo receptors.
    Elias Adriaenssens, Luca Ferrari, Sascha Martens.
      Cellular homeostasis requires the swift and specific removal of damaged material. Selective autophagy represents a major pathway for the degradation of such cargo material. This is achieved by the sequestration of the cargo within double-membrane vesicles termed autophagosomes, which form de novo around the cargo and subsequently deliver their content to lysosomes for degradation. The importance of selective autophagy is exemplified by the various neurodegenerative diseases associated with defects in this pathway, including Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia. It has become evident that cargo receptors are acting as Swiss army knives in selective autophagy by recognizing the cargo, orchestrating the recruitment of the machinery for autophagosome biogenesis, and closely aligning the membrane with the cargo. Furthermore, cargo receptors sequester ubiquitinated proteins into larger condensates upstream of autophagy induction. Here, we review recent insights into the mechanisms of action of cargo receptors in selective autophagy by focusing on the roles of sequestosome-like cargo receptors in the degradation of misfolded, ubiquitinated proteins and damaged mitochondria. We also highlight at which steps defects in their function result in the accumulation of harmful material and how this knowledge may guide the design of future therapies.
    DOI:  https://doi.org/10.1016/j.cub.2022.11.002
  13. RSC Med Chem. 2022 Dec 14. 13(12): 1476-1494
    Targeted protein degradation using the lysosomal pathway.
    Samrajni Ghosh, Bhavana Ramadas, Debasish Manna.
      Degradation strategies have shown enormous promise after the inception of molecules like PROTACs (PRoteolysis TArgeting Chimeras) that induce the degradation of the substrate of choice rather than depending on blocking their catalytic activity like conventional inhibitory drugs. Over the past two decades, the application of PROTACs has made quite an impact, even reaching clinical translations. However, a major class of macromolecular targets, be that large proteins, aggregates, organelles or non-protein substrates, remain untouched when utilizing the ubiquitin-proteasomal pathway of degradation. In this review, we have attempted to cover modalities of targeted degradation that instead focus on recruiting the lysosomal pathway of degradation, which is gaining importance and being explored extensively as alternate and efficient approaches for treating disease-related milieus.
    DOI:  https://doi.org/10.1039/d2md00273f
  14. Sci Adv. 2022 Dec 23. 8(51): eadd3942
    Regulation of eIF4E guides a unique translational program to control erythroid maturation.
    Craig M Forester, Juan A Oses-Prieto, Nancy J Phillips, Sohit Miglani, Xiaming Pang, Gun Woo Byeon, Rachel DeMarco, Al Burlingame, Maria Barna, Davide Ruggero.
      Translation control is essential in balancing hematopoietic precursors and differentiation; however, the mechanisms underlying this program are poorly understood. We found that the activity of the major cap-binding protein eIF4E is unexpectedly regulated in a dynamic manner throughout erythropoiesis that is uncoupled from global protein synthesis rates. Moreover, eIF4E activity directs erythroid maturation, and increased eIF4E expression maintains cells in an early erythroid state associated with a translation program driving the expression of PTPN6 and Igf2bp1. A cytosine-enriched motif in the 5' untranslated region is important for eIF4E-mediated translation specificity. Therefore, selective translation of key target genes necessary for the maintenance of early erythroid states by eIF4E highlights a unique mechanism used by hematopoietic precursors to rapidly elicit erythropoietic maturation upon need.
    DOI:  https://doi.org/10.1126/sciadv.add3942
  15. Elife. 2022 Dec 22. pii: e69162. [Epub ahead of print]11
    Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia.
    Anne-Claire Godet, Emilie Roussel, Florian P David, Fransky Hantelys, Florent Morfoisse, Joffrey Alves, Françoise Pujol, Isabelle Ader, Edouard Bertrand, Odile Burlet-Schiltz, Carine Froment, Anthony K Henras, Patrice Vitali, Eric Lacazette, Florence Tatin, Barbara Garmy-Susini, Anne-Catherine Prats.
      Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and RPS2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response.
    Keywords:  cell biology; chromosomes; gene expression; mouse
    DOI:  https://doi.org/10.7554/eLife.69162
  16. Am J Pathol. 2022 Dec 16. pii: S0002-9440(22)00397-2. [Epub ahead of print]
    Mitochondria and Endoplasmic Reticulum Stress in Retinal Organoids from Vision Loss Patients.
    Eun-Jin Lee, Monica Sophia Diaz-Aguilar, Hyejung Min, Jihee Choi, Diego Adrian Valdez Duran, Julia M Grandjean, R Luke Wiseman, Heike Kroeger, Jonathan H Lin.
      Activating transcription factor 6 (ATF6), a key regulator of the unfolded protein response (UPR), is required for endoplasmic reticulum (ER) function and protein homeostasis. Variants of ATF6 that abrogate transcriptional activity cause morphologic and molecular defects in cones manifesting clinically as the human vision loss disease achromatopsia (ACHM). ATF6 is expressed in all retinal cells. However, the effect of disease-associated ATF6 variants on other retinal cell types remains unclear. To investigate this question, we analyzed bulk-RNA-seq transcriptomes from retinal-organoids generated from ACHM patients carrying homozygous loss-of-function ATF6 variants. We identified marked dysregulation in mitochondrial respiratory complex gene expression and disrupted mitochondrial morphology in ACHM retinal organoids, indicating that loss of ATF6 leads to previously unappreciated mitochondrial defects in the retina. Next, we compared gene expression from control and ACHM retinal organoids with transcriptome profiles of 7 major retinal cell types generated from recent single-cell transcriptomic maps of non-diseased human retina. Our analysis revealed pronounced down-regulation of cone genes and up-regulation in Müller glia genes, with no significant effects on other retinal cells. Overall, our analysis of ACHM patient retinal organoids identifies new cellular and molecular phenotypes in addition to cone dysfunction: activation of Müller cells, increased ER stress, and disrupted mitochondrial structure and elevated respiratory chain activity gene expression.
    DOI:  https://doi.org/10.1016/j.ajpath.2022.12.002
  17. FEBS J. 2022 Dec 19.
    Deregulation of HSF1-mediated endoplasmic reticulum unfolded protein response promotes cisplatin resistance in lung cancer cells.
    Min Zhang, Xiaoyu Duan, Lu Wang, Jing Wen, Pingfei Fang.
      Mild hypothermia can induce apoptotic cell death in many cancer cells, but the underlying mechanisms remain unclear. In a genetic screen in C. elegans, we found that impaired endoplasmic reticulum unfolded protein response (UPRER ) increased animal survival after cold shock. Consistently, in normal human lung cells, decreasing culture temperature from 37°C to 30°C activated UPRER and promoted cell death. However, lung adenocarcinoma cells were impaired in UPRER induction and resistant to hypothermia-induced cell death. Mechanistically, hypothermic stress increased HSF1 levels, which in turn activated UPRER to promote apoptotic cell death. HSF1 expression was associated with UPRER genes in normal tissues but such association was lost in many cancers, especially lung adenocarcinoma. Activating UPRER enhanced the cytotoxicity of chemotherapy drugs cisplatin preferentially in cancer cells. Consistently, cancer patients with higher UPRER expression had generally better prognosis. Together, our study on hypothermia has led to the discovery of HSF1-UPRER in the regulation of drug sensitivity in lung cancer cells, providing novel thoughts on developing new strategies against chemoresistance.
    Keywords:  HSF1; chemoresistance; hypothermia; lung cancer; unfolded protein response
    DOI:  https://doi.org/10.1111/febs.16709
  18. Bioorg Med Chem Lett. 2022 Dec 19. pii: S0960-894X(22)00587-X. [Epub ahead of print] 129111
    Recent Advances toward the Development of Hsp90 C-terminal Inhibitors.
    Eva Amatya, Brian S J Blagg.
      Heat shock protein 90 (Hsp90) is a dynamic protein which serves to ensure proper folding of nascent client proteins, regulate transcriptional responses to environmental stress and guide misfolded and damaged proteins to destruction via ubiquitin proteasome pathway. Recent advances in the field of Hsp90 have been made through development of isoform selective inhibitors, Hsp90 C-terminal inhibitors and disruption of protein-protein interactions. These approaches have led to alleviation of adverse off-target effects caused by pan-inhibition of Hsp90 using N-terminal inhibitors. In this review, we provide an overview of relevant advances on targeting the Hsp90 C-terminal Domain (CTD) and the development of Hsp90 C-terminal inhibitors (CTIs) since 2015. The proper functioning and folding of proteins is necessary for cell survival.1 Cellular stresses such as acidosis, exposure to toxins, viruses, DNA damage, metabolic and oxidative stress, and/or hypoxia lead to the disruption of proteostasis, and can result in a lethal outcome. To maintain proteostasis upon exposure to cellular stresses, cells induce the expression of molecular chaperones such as the heat shock proteins.2 This protein family is divided based on molecular weight and includes Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, as well as other larger members, each of which plays a unique role during the protein folding cycle.3 The 90kDa Heat Shock Proteins (Hsp90) are ubiquitous and highly-conserved molecular chaperones that are responsible for the activation, stabilization and maturation of ∼400 client protein substrates. Hsp90 comprises ∼1-2% of all cellular proteins, however, Hsp90 expression is elevated to ∼4-6% of total protein content in cancer cells. Hsp90 is a homodimer with each monomer consisting of three components; an ATP-binding N-terminal domain (NTD), a middle domain (MD) wherein protein-protein interactions occur, and a C-terminal domain (CTD) that is responsible for dimerization.4 The CTD contains a nucleotide binding site that allosterically modulates ATPase activity within the NTD.5 The CTD also possesses a conserved Met-Glu-Glu-Val-Asp (MEEVD) sequence at the terminus to provide interactions with co-chaperones that contain a tetratricopeptide-containing repeat (TPR).6 These interactions with co-chaperones are vital for the regulation and progression of the Hsp90 protein folding cycle.
    Keywords:  Anti-cancer; Chaperones; Hasp90; Neuroprotective; Novobiocin
    DOI:  https://doi.org/10.1016/j.bmcl.2022.129111
  19. Apoptosis. 2022 Dec 19.
    The unfolded protein response (UPR) pathway: the unsung hero in breast cancer management.
    Arunita Patra, Arghya Adhikary, Swatilekha Ghosh.
      Tumor cells always have the need to produce an increased amount of proteins in the cells. This elevated amount of proteins increases the pressure on the organelles of the cell such as the endoplasmic reticulum and compels it to increase its protein folding efficiency. However, it is by a matter of fact, that the amount of proteins synthesized outweighs the protein folding capacity of the ER which in turn switches on the UPR pathway by activating the three major molecular sensors and other signaling cascades, which helps in cell survival instead of instant death. However, if this pathway is active for a prolonged period of time the tumor cells heads toward apoptosis. Again, interestingly this is not the same as in case of non- tumorogenic cells. This exhibit a straight natural pathway for tumor cells-specific destruction which has a great implication in today's world where hormone therapies and chemo-therapies are non-effective for various types of breast cancer, a major type being Triple Negative Breast Cancer. Thus a detailed elucidation of the molecular involvement of the UPR pathway in breast cancer may open new avenues for management and attract novel chemotherapeutic targets providing better hopes to patients worldwide.
    Keywords:  Apoptosis; Breast cancer; ER Stress; GRP78; UPR sensors
    DOI:  https://doi.org/10.1007/s10495-022-01803-3
  20. Science. 2022 Dec 20. eabo3627
    Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children.
    CoV-Contact Cohort§
      Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic dsRNA-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the ssRNA-degrading RNase L. Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNASEL deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or SARS-CoV-2 stimulation. Exogenous 2-5A suppresses cytokine production in OAS1- but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by MAVS deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.
    DOI:  https://doi.org/10.1126/science.abo3627
  21. Commun Biol. 2022 Dec 22. 5(1): 1402
    Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays.
    Ehsan Manouchehri Doulabi, Claudia Fredolini, Radiosa Gallini, Liza Löf, Qiujin Shen, Ryoyo Ikebuchi, Louise Dubois, Alireza Azimi, Olivier Loudig, Susanne Gabrielsson, Ulf Landegren, Anders Larsson, Jonas Bergquist, Masood Kamali-Moghaddam.
      Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and  proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs' surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA.
    DOI:  https://doi.org/10.1038/s42003-022-04349-x
  22. FEBS Lett. 2022 Dec 17.
    Does the nuclear envelope retain its identity during mitosis?
    Natsumi Tsuda, Ryohei Fukagawa, Shinji Sueda.
      During mitosis in metazoan species, the nuclear envelope (NE) undergoes breakdown, and its fragments are absorbed within the membranous network of the endoplasmic reticulum (ER). Past observations by fluorescence microscopy led researchers to think that the NE loses its identity when it is absorbed within the ER membrane. However, in our previous work, we developed a more specific labeling method and found evidence that the NE does not completely lose its identity during mitosis. In the present work, we conduct further experiments, the results of which support the idea that the NE partially retains its identity during mitosis.
    Keywords:  NE breakdown; NE marker; NE reformation; biotinylation; fluorescent labeling; mitosis; nuclear envelope
    DOI:  https://doi.org/10.1002/1873-3468.14568
  23. Traffic. 2022 Dec 23.
    Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.
    Cristiana Lungu, Florian Meyer, Marcel Hörning, Jasmin Steudle, Anja Braun, Bettina Noll, David Benz, Felix Fränkle, Simone Schmid, Stephan A Eisler, Monilola A Olayioye.
      The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen we here identify the RhoGEF protein Solo as a functional counterpart of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.
    DOI:  https://doi.org/10.1111/tra.12880
  24. J Biol Chem. 2022 Dec 15. pii: S0021-9258(22)01249-2. [Epub ahead of print] 102806
    A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity.
    Charlotte M Fare, Kevin Rhine, Andrew Lam, Sua Myong, James Shorter.
      Karyopherin-β2 (Kapβ2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals (PY-NLSs) of diverse cytoplasmic cargo for transport to the nucleus. Kapβ2 cargo include several disease-linked RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RBPs with PrLDs are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and multisystem proteinopathy (MSP). Remarkably, Kapβ2 prevents and reverses aberrant phase transitions of these cargo, which is cytoprotective. However, the molecular determinants of Kapβ2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapβ2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapβ2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8-20 of Kapβ2 recapitulate all salient features of Kapβ2 activity. By contrast, Kapβ2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapβ2 construct for delivery in adeno-associated viruses as a potential therapeutic for ALS/FTD, MSP, and related disorders.
    Keywords:  FUS; chaperone; disaggregase; nuclear-import receptor; nucleocytoplasmic transport; phase separation
    DOI:  https://doi.org/10.1016/j.jbc.2022.102806
  25. Development. 2022 Dec 15. pii: dev201213. [Epub ahead of print]149(24):
    Translational control by maternal Nanog promotes oogenesis and early embryonic development.
    Mudan He, Shengbo Jiao, Ru Zhang, Ding Ye, Houpeng Wang, Yonghua Sun.
      Many maternal mRNAs are translationally repressed during oocyte development and spatio-temporally activated during early embryogenesis, which is crucial for oocyte and early embryo development. By analyzing maternal mutants of nanog (Mnanog) in zebrafish, we demonstrated that Nanog tightly controls translation of maternal mRNA during oogenesis via transcriptional repression of eukaryotic translation elongation factor 1 alpha 1, like 2 (eef1a1l2). Loss of maternal Nanog led to defects of egg maturation, increased endoplasmic reticulum stress, and an activated unfold protein response, which was caused by elevated translational activity. We further demonstrated that Nanog, as a transcriptional repressor, represses the transcription of eefl1a1l2 by directly binding to the eef1a1l2 promoter in oocytes. More importantly, depletion of eef1a1l2 in nanog mutant females effectively rescued the elevated translational activity in oocytes, oogenesis defects and embryonic defects of Mnanog embryos. Thus, our study demonstrates that maternal Nanog regulates oogenesis and early embryogenesis through translational control of maternal mRNA via a mechanism whereby Nanog acts as a transcriptional repressor to suppress transcription of eef1a1l2.
    Keywords:  Embryonic development; Nanog; Oogenesis; Translational control; Zebrafish
    DOI:  https://doi.org/10.1242/dev.201213
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