bims-proteo Biomed News
on Proteostasis
Issue of 2021‒01‒03
sixty papers selected by
Eric Chevet
INSERM


  1. Proc Natl Acad Sci U S A. 2021 Jan 05. pii: e2011763118. [Epub ahead of print]118(1):
    Balce DR, Wang YT, McAllaster MR, Dunlap BF, Orvedahl A, Hykes BL, Droit L, Handley SA, Wilen CB, Doench JG, Orchard RC, Stallings CL, Virgin HW.
      Macrophages activated with interferon-γ (IFN-γ) in combination with other proinflammatory stimuli, such as lipopolysaccharide or tumor necrosis factor-α (TNF-α), respond with transcriptional and cellular changes that enhance clearance of intracellular pathogens at the risk of damaging tissues. IFN-γ effects must therefore be carefully balanced with inhibitory mechanisms to prevent immunopathology. We performed a genome-wide CRISPR knockout screen in a macrophage cell line to identify negative regulators of IFN-γ responses. We discovered an unexpected role of the ubiquitin-fold modifier (Ufm1) conjugation system (herein UFMylation) in inhibiting responses to IFN-γ and lipopolysaccharide. Enhanced IFN-γ activation in UFMylation-deficient cells resulted in increased transcriptional responses to IFN-γ in a manner dependent on endoplasmic reticulum stress responses involving Ern1 and Xbp1. Furthermore, UFMylation in myeloid cells is required for resistance to influenza infection in mice, indicating that this pathway modulates in vivo responses to infection. These findings provide a genetic roadmap for the regulation of responses to a key mediator of cellular immunity and identify a molecular link between the UFMylation pathway and immune responses.
    Keywords:  ER stress; UFMylation; autophagy; immunology; interferon
    DOI:  https://doi.org/10.1073/pnas.2011763118
  2. J Biol Chem. 2020 Dec 30. pii: jbc.RA120.015162. [Epub ahead of print]
    Harris LD, Le Pen J, Scholz N, Mieszczanek J, Vaughan N, Davis S, Berridge G, Kessler B, Bienz M, Licchesi JDF.
      Ubiquitin is a versatile post-translational modification which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains which have been extensively studied, the regulation and function of most atypical ubiquitin chains is only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. We then validated the E3 ubiquitin ligase HECTD1 as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knock out of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase which assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.
    Keywords:  E3 ubiquitin ligase; HECTD1; K29/K48-linked polyubiquitin chain; TRABID; branched ubiquitin chains; deubiquitylation (deubiquitination); polyubiquitin chain; protein degradation; protein-protein interaction; proteomics; ubiquitin; ubiquitin ligase; ubiquitin thioesterase (OTUB1)
    DOI:  https://doi.org/10.1074/jbc.RA120.015162
  3. Science. 2021 Jan 01. pii: eabb6896. [Epub ahead of print]371(6524):
    You K, Wang L, Chou CH, Liu K, Nakata T, Jaiswal A, Yao J, Lefkovith A, Omar A, Perrigoue JG, Towne JE, Regev A, Graham DB, Xavier RJ.
      Tissue homeostasis is perturbed in a diversity of inflammatory pathologies. These changes can elicit endoplasmic reticulum (ER) stress, protein misfolding, and cell death. ER stress triggers the unfolded protein response (UPR), which can promote recovery of ER proteostasis and cell survival or trigger programmed cell death. Here, we leveraged single-cell RNA sequencing to define dynamic transcriptional states associated with the adaptive versus terminal UPR in the mouse intestinal epithelium. We integrated these transcriptional programs with genome-scale CRISPR screening to dissect the UPR pathway functionally. We identified QRICH1 as a key effector of the PERK-eIF2α axis of the UPR. QRICH1 controlled a transcriptional program associated with translation and secretory networks that were specifically up-regulated in inflammatory pathologies. Thus, QRICH1 dictates cell fate in response to pathological ER stress.
    DOI:  https://doi.org/10.1126/science.abb6896
  4. Hum Mol Genet. 2020 Nov 06. pii: ddaa243. [Epub ahead of print]
    Cantarero L, Juárez-Escoto E, Civera-Tregón A, Rodríguez-Sanz M, Roldán M, Benítez R, Hoenicka J, Palau F.
      Mutations in the GDAP1 gene cause Charcot-Marie-Tooth (CMT) neuropathy. GDAP1 is an atypical glutathione S-transferase (GST) of the outer mitochondrial membrane and the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs). Here, we investigate the role of this GST in the autophagic flux and the membrane contact sites (MCSs) between mitochondria and lysosomes in the cellular pathophysiology of GDAP1 deficiency. We demonstrate that GDAP1 participates in basal autophagy and that its depletion affects LC3 and PI3P biology in autophagosome biogenesis and membrane trafficking from MAMs. GDAP1 also contributes to the maturation of lysosome by interacting with PYKfyve kinase, a pH-dependent master lysosomal regulator. GDAP1 deficiency causes giant lysosomes with hydrolytic activity, a delay in the autophagic lysosome reformation, and TFEB activation. Notably, we found that GDAP1 interacts with LAMP-1, which supports that GDAP1-LAMP-1 is a new tethering pair of mitochondria and lysosome membrane contacts. We observed mitochondria-lysosome MCSs in soma and axons of cultured mouse embryonic motor neurons and human neuroblastoma cells. GDAP1 deficiency reduces the MCSs between these organelles, causes mitochondrial network abnormalities, and decreases levels of cellular glutathione (GSH). The supply of GSH-MEE suffices to rescue the lysosome membranes and the defects of the mitochondrial network, but not the interorganelle MCSs nor early autophagic events. Overall, we show that GDAP1 enables the proper function of mitochondrial MCSs in both degradative and nondegradative pathways, which could explain primary insults in GDAP1-related CMT pathophysiology, and highlights new redox-sensitive targets in axonopathies where mitochondria and lysosomes are involved.
    DOI:  https://doi.org/10.1093/hmg/ddaa243
  5. Life (Basel). 2020 Dec 22. pii: E1. [Epub ahead of print]11(1):
    Aghaei M, Dastghaib S, Aftabi S, Aghanoori MR, Alizadeh J, Mokarram P, Mehrbod P, Ashrafizadeh M, Zarrabi A, McAlinden KD, Eapen MS, Sohal SS, Sharma P, Zeki AA, Ghavami S.
      Cellular protein homeostasis in the lungs is constantly disrupted by recurrent exposure to various external and internal stressors, which may cause considerable protein secretion pressure on the endoplasmic reticulum (ER), resulting in the survival and differentiation of these cell types to meet the increased functional demands. Cells are able to induce a highly conserved adaptive mechanism, known as the unfolded protein response (UPR), to manage such stresses. UPR dysregulation and ER stress are involved in numerous human illnesses, such as metabolic syndrome, fibrotic diseases, and neurodegeneration, and cancer. Therefore, effective and specific compounds targeting the UPR pathway are being considered as potential therapies. This review focuses on the impact of both external and internal stressors on the ER in idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) and discusses the role of the UPR signaling pathway activation in the control of cellular damage and specifically highlights the potential involvement of non-coding RNAs in COPD. Summaries of pathogenic mechanisms associated with the ER stress/UPR axis contributing to IPF and COPD, and promising pharmacological intervention strategies, are also presented.
    Keywords:  UPR; autophagy; endoplasmic reticulum; fibrosis; lung disease; non-coding RNA; tissue remodeling
    DOI:  https://doi.org/10.3390/life11010001
  6. Cell Rep. 2020 Dec 29. pii: S2211-1247(20)31552-7. [Epub ahead of print]33(13): 108563
    Li X, Sun S, Appathurai S, Sundaram A, Plumb R, Mariappan M.
      Misfolded proteins in the endoplasmic reticulum (ER) activate IRE1α endoribonuclease in mammalian cells, which mediates XBP1 mRNA splicing to produce an active transcription factor. This promotes the expression of specific genes to alleviate ER stress, thereby attenuating IRE1α. Although sustained activation of IRE1α is linked to human diseases, it is not clear how IRE1α is attenuated during ER stress. Here, we identify that Sec63 is a subunit of the previously identified IRE1α/Sec61 translocon complex. We find that Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto IRE1α. This leads to inhibition of higher-order oligomerization and attenuation of IRE1α RNase activity during prolonged ER stress. In Sec63-deficient cells, IRE1α remains activated for a long period of time despite the presence of excess BiP in the ER. Thus, our data suggest that the Sec61 translocon bridges IRE1α with Sec63/BiP to regulate the dynamics of IRE1α signaling in cells.
    Keywords:  ER stress; IRE1; Sec61 translocon; endoplasmic reticulum; protein translocation; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2020.108563
  7. Biochim Biophys Acta Gen Subj. 2020 Dec 25. pii: S0304-4165(20)30345-7. [Epub ahead of print]1865(3): 129834
    Kim KH, Lee MS.
      BACKGROUND: Mitochondria is a key organelle for energy production and cellular adaptive response to intracellular and extracellular stresses. Mitochondrial stress can be evoked by various stimuli such as metabolic stressors or pathogen infection, which may lead to expression of 'mitokines' such as growth differentiation factor 15 (GDF15).SCOPE OF REVIEW: This review summarizes the mechanism of GDF15 expression in response to organelle stress such as mitochondrial stress, and covers pathophysiological conditions or diseases that are associated with elevated GDF15 level. This review also illustrates the in vivo role of GDF15 expression in those stress conditions or diseases, and a potential of GDF15 as a therapeutic agent against metabolic disorders such as NASH.
    MAJOR CONCLUSIONS: Mitochondrial unfolded protein response (UPRmt) is a critical process to recover from mitochondrial stress. UPRmt can induce expression of secretory proteins that can exert systemic effects (mitokines) as well as mitochondrial chaperons. GDF15 can have either protective or detrimental systemic effects in response to mitochondrial stresses, suggesting its role as a mitokine. Mounting evidence shows that GDF15 is also induced by stresses of organelles other than mitochondria such as endoplasmic reticulum (ER). GDF15 level is increased in serum or tissue of mice and human subjects with metabolic diseases such as obesity or NASH. GDF15 can modulate metabolic features of those diseases.
    GENERAL SIGNIFICANCE: GDF15 play a role as an integrated stress response (ISR) beyond mitochondrial stress response. GDF15 is involved in the pathogenesis of metabolic diseases such as NASH, and also could be a candidate for therapeutic agent against those diseases.
    Keywords:  ER; GDF15; Metabolic diseases; Mitochondria; NASH; Stress
    DOI:  https://doi.org/10.1016/j.bbagen.2020.129834
  8. Curr Genet. 2021 Jan 01.
    Boronat S, Cabrera M, Hidalgo E.
      Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another. Hence thermo-sensitive proteins that lose their active conformation might be refolded with the aid of chaperones or removed by the ubiquitin-proteasome system or the process of autophagy. We have recently developed thermo-sensitive reporters to study PQC in fission yeast and shown the relevance of a third adaptation strategy: the sequestration of misfolded proteins into inclusions which will prevent a rapid degradation and allow the refolding once stress ends. These protein inclusions, protein aggregate centers (PACs), contain a broad spectrum of misfolding/aggregation-prone proteins and chaperones involved in their assembly or dissolution. The chaperone couple Mas5/Ssa2 plays a crucial role in PAC formation, whereas the Hsp104 chaperone promotes their disassembly. The absence of aggregates observed in cells lacking Mas5 could be also explained by the activation of the transcription factor Hsf1 and the induction of chaperone genes, we have excluded this possibility here demonstrating that increased Hsf1 activity and the subsequent overexpression of chaperones do not prevent the assembly of protein aggregates. Protein deposition at certain locations also constitutes a tactic to inactivate proteins temporally. This is the case of Pyp1, the main phosphatase of the stress response kinase Sty1. Upon stress imposition, misfolded Pyp1 is sequestered into cytosolic protein foci while active Sty1 at the nucleus switches on the transcriptional response. In conclusion, we propose that the assembly of aggregation-like foci, PACs in fission yeast, is a crucial PQC strategy during heat stress, and that the Hsp40 chaperone Mas5 is required for PAC assembly and connects physiological and heat-shock triggered PQC.
    Keywords:  Heat stress; Hsp40; Mas5; PAC; PQC; Protein aggregate centers; Pyp1; Sty1
    DOI:  https://doi.org/10.1007/s00294-020-01135-2
  9. EMBO Rep. 2020 Dec 28. e50163
    Roedig J, Kowald L, Juretschke T, Karlowitz R, Ahangarian Abhari B, Roedig H, Fulda S, Beli P, van Wijk SJ.
      Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin-specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono-ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFα-mediated NF-κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor-interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22-regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis-associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death.
    Keywords:  RIPK1; cancer; mixed lineage kinase domain-like; post-translational modifications; ubiquitin hydrolase
    DOI:  https://doi.org/10.15252/embr.202050163
  10. Int J Mol Sci. 2020 Dec 23. pii: E85. [Epub ahead of print]22(1):
    Sanwald JL, Dobner J, Simons IM, Poschmann G, Stühler K, Üffing A, Hoffmann S, Willbold D.
      GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors SNAREs was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome compositionofTKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.
    Keywords:  Atg8; GABARAP; Golgi apparatus; surfaceome
    DOI:  https://doi.org/10.3390/ijms22010085
  11. J Biol Chem. 2020 Dec 23. pii: jbc.RA120.016858. [Epub ahead of print]
    Wu K, Ching K, Chong RA, Pan ZQ.
      Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence based techniques in ubiquitination studies remain underexplored at least partly due to challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, Förster resonance energy transfer (FRET) di-ubiquitination, to track substrate ubiquitination by fluorescence.This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The di-ubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET di-ubiquitination relies on Ub-substrate fusion, which can be implemented using either one of the two validated strategies. Method one is the use of recombinant substrate-Ub fusion, applicable to all substrate peptides that can bind to E3. Method two is a chemo-enzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based di-ubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.
    Keywords:  E3 ubiquitin ligase; chemo-enzymatic ligation; fluorescence resonance energy transfer (FRET); kinetics; protein degradation; ubiquitylation (ubiquitination)
    DOI:  https://doi.org/10.1074/jbc.RA120.016858
  12. J Cell Physiol. 2020 Dec 23.
    Jin YG, Zhou H, Fan D, Che Y, Wang ZP, Wang SS, Tang QZ.
      TMEM173 has been reported to participate in endoplasmic reticulum stress, inflammation and immunology, all of which closely involved with cardiac hypertrophy. But its role in autophagy is not fully figured out. In our research, Tmem173 global knockout (KO) mice manifested more deteriorated hypertrophy, fibrosis, inflammatory infiltration and cardiac malfunction compared with wild type C57BL/6 mice after 6 weeks of transverse aortic constriction. And KO mice showed inhibited autophagosome degradation in myocardium observed under transmission electron microscope and in protein level. In in vitro experiments conducted in neonatal rat cardiomyocytes under phenylephrine treatment, the abundance of Tmem173 gene was negatively related to the abundance of LC3-Ⅱ and the number of red and yellow fluorescent dots, of which reflected the capacity of autophagosome degradation. These results indicated that TMEM173 might be a promoter of autophagic flux and protected against pressure overload-induced cardiac hypertrophy. It may serve as a potential therapeutic target for cardiac hypertrophy in the future.
    Keywords:  TMEM173; ULK1; autophagy; cardiac hypertrophy; fibrosis
    DOI:  https://doi.org/10.1002/jcp.30223
  13. J Cell Biol. 2021 Mar 01. pii: e201912041. [Epub ahead of print]220(3):
    Work JJ, Brandman O.
      Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.
    DOI:  https://doi.org/10.1083/jcb.201912041
  14. PLoS Pathog. 2020 Dec 23. 16(12): e1008504
    Mohanty S, Han T, Choi YB, Lavorgna A, Zhang J, Harhaj EW.
      Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.
    DOI:  https://doi.org/10.1371/journal.ppat.1008504
  15. Cell Struct Funct. 2020 Dec 22.
    Yaguchi K, Sato K, Yoshizawa K, Mikami D, Yuyama K, Igarashi Y, Banhegyi G, Margittai E, Uehara R.
      The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control. Key words: Haploid, ER stress, Mevalonate, pathway.
    Keywords:  ER stress; Haploid; Mevalonate; pathway
    DOI:  https://doi.org/10.1247/csf.20055
  16. PLoS Comput Biol. 2020 Dec 28. 16(12): e1008492
    Mallela A, Nariya MK, Deeds EJ.
      Protein turnover is vital to cellular homeostasis. Many proteins are degraded efficiently only after they have been post-translationally "tagged" with a polyubiquitin chain. Ubiquitylation is a form of Post-Translational Modification (PTM): addition of a ubiquitin to the chain is catalyzed by E3 ligases, and removal of ubiquitin is catalyzed by a De-UBiquitylating enzyme (DUB). Nearly four decades ago, Goldbeter and Koshland discovered that reversible PTM cycles function like on-off switches when the substrates are at saturating concentrations. Although this finding has had profound implications for the understanding of switch-like behavior in biochemical networks, the general behavior of PTM cycles subject to synthesis and degradation has not been studied. Using a mathematical modeling approach, we found that simply introducing protein turnover to a standard modification cycle has profound effects, including significantly reducing the switch-like nature of the response. Our findings suggest that many classic results on PTM cycles may not hold in vivo where protein turnover is ubiquitous. We also found that proteins sharing an E3 ligase can have closely related changes in their expression levels. These results imply that it may be difficult to interpret experimental results obtained from either overexpressing or knocking down protein levels, since changes in protein expression can be coupled via E3 ligase crosstalk. Understanding crosstalk and competition for E3 ligases will be key in ultimately developing a global picture of protein homeostasis.
    DOI:  https://doi.org/10.1371/journal.pcbi.1008492
  17. Curr Opin Virol. 2020 Dec 21. pii: S1879-6257(20)30111-5. [Epub ahead of print]47 9-17
    Verchot J, Pajerowska-Mukhtar KM.
      In recent years there have been significant advances in our understanding of the ER stress responses in plants that are associated with virus infection, as well as bacterial and fungal diseases. In plants, ER stress induced by virus infection includes several signaling pathways that include the unfolded protein response (UPR) to promote the expression of chaperone proteins for proper protein folding. Understanding how facets of ER stress signaling broadly engage in pathogen responses, as well as those that are specific to virus infection is important to distinguishing features essential for broad cellular defenses and processes that may be specifically linked to viral infectivity and disease.
    DOI:  https://doi.org/10.1016/j.coviro.2020.11.001
  18. Mol Cell. 2020 Dec 17. pii: S1097-2765(20)30837-6. [Epub ahead of print]
    Kraushar ML, Krupp F, Harnett D, Turko P, Ambrozkiewicz MC, Sprink T, Imami K, Günnigmann M, Zinnall U, Vieira-Vieira CH, Schaub T, Münster-Wandowski A, Bürger J, Borisova E, Yamamoto H, Rasin MR, Ohler U, Beule D, Mielke T, Tarabykin V, Landthaler M, Kramer G, Vida I, Selbach M, Spahn CMT.
      Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.
    Keywords:  Ebp1; cryo-EM; development; neocortex; neurons; pSILAC/BONCAT mass spectrometry; proteostasis; ribosome; ribosome profiling; selective ribosome profiling
    DOI:  https://doi.org/10.1016/j.molcel.2020.11.037
  19. Biochem Biophys Res Commun. 2020 Dec 24. pii: S0006-291X(20)32205-1. [Epub ahead of print]536 52-58
    Nakao H, Seko A, Ito Y, Sakono M.
      The lectin chaperones calnexin (CNX) and calreticulin (CRT) localized in the endoplasmic reticulum play important roles in glycoprotein quality control. Although the interaction between these lectin chaperones and ERp57 is well known, it has been recently reported that endoplasmic reticulum protein 29 (ERp29), a member of PDI family, interacts with CNX and CRT. The biochemical function of ERp29 is unclear because it exhibits no ERp57-like redox activity. In this study, we addressed the possibility that ER chaperones CNX and CRT are connected via ERp29, based on our observation that ERp29 exists as a dimer. As a result, we showed that CNX dimerizes through ERp29. These results endorse the hypothesis that ERp29 serves as a bridge that links two molecules of CNX. Also, we showed that similar complexes such as CNX-CRT were formed via ERp29.
    Keywords:  Calnexin; Calreticulin; Endoplasmic reticulum; Endoplasmic reticulum protein 29; Molecular chaperone
    DOI:  https://doi.org/10.1016/j.bbrc.2020.12.023
  20. Biochem J. 2020 Dec 23. pii: BCJ20200885. [Epub ahead of print]
    Ikeda M, Watanabe T, Ito A, Fujimuro M.
      Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Further, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.
    Keywords:  UBE1; herpesvirus; post translational modification; ubiquitin proteasome system; ubiquitins; virus replication
    DOI:  https://doi.org/10.1042/BCJ20200885
  21. Clin Sci (Lond). 2020 Dec 23. pii: CS20201137. [Epub ahead of print]
    Delgado-Valero B, de la Fuente-Chávez L, Romero-Miranda A, Vistación Bartolomé M, Ramachandi B, Islas F, Luaces M, Cachofeiro V, Martinez-Martinez E.
      Myocardial infarction (MI) is associated with renal alterations resulting in poor outcomes in patients with MI. Renal fibrosis is a potent predictor of progression in patients and is often accompanied by inflammation and oxidative stress; however, the mechanisms involved in these alterations are not well established. Endoplasmic reticulum (ER) plays a central role in protein processing and folding. An accumulation of unfolded proteins leads to ER dysfunction, termed ER stress. Since the kidney is the organ with highest protein synthesis fractional rate, we herein investigated the effects of MI on ER stress at renal level, as well as the possible role of ER stress on renal alterations after MI. Patients and MI male Wistar rats showed an increase in the kidney injury marker neutrophil gelatinase-associated lipocalin (NGAL) at circulating level. Four weeks post-MI rats presented renal fibrosis, oxidative stress and inflammation accompanied by ER stress activation characterized by enhanced immunoglobin binding protein (BiP), protein disulfide-isomerase A6 (PDIA6) and activating transcription factor 6-alpha (ATF6α) protein levels. In renal fibroblasts, palmitic acid (PA; 50-200 µM) and angiotensin II (Ang II; 10-8-10-6M) promoted extracellular matrix, superoxide anion production and inflammatory markers upregulation. The presence of the ER stress inhibitor, 4-phenylbutyric acid (4-PBA; 4 µM), was able to prevent all of these modifications in renal cells. Therefore, the data show that ER stress mediates the deleterious effects of PA and Ang II in renal cells and support the potential role of ER stress on renal alterations associated with MI.
    Keywords:  endoplasmic reticulum stress; myocardial infarction; renal fibrosis
    DOI:  https://doi.org/10.1042/CS20201137
  22. Cell Rep. 2020 Dec 22. pii: S2211-1247(20)31533-3. [Epub ahead of print]33(12): 108544
    Rong B, Zhang Q, Wan J, Xing S, Dai R, Li Y, Cai J, Xie J, Song Y, Chen J, Zhang L, Yan G, Zhang W, Gao H, Han JJ, Qu Q, Ma H, Tian Y, Lan F.
      N6 methylation at adenosine 1832 (m6A1832) of mammalian 18S rRNA, occupying a critical position within the decoding center, is modified by a conserved methyltransferase, METTL5. Here, we find that METTL5 shows strong substrate preference toward the 18S A1832 motif but not the other reported m6A motifs. Comparison with a yeast ribosome structural model unmodified at this site indicates that the modification may facilitate mRNA binding by inducing conformation changes in the mammalian ribosomal decoding center. METTL5 promotes p70-S6K activation and proper translation initiation, and the loss of METTL5 significantly reduces the abundance of polysome. METTL5 expression is elevated in breast cancer patient samples and is required for growth of several breast cancer cell lines. We further find that Caenorhabditis elegans lacking the homolog metl-5 develop phenotypes known to be associated with impaired translation. Altogether, our findings uncover critical and conserved roles of METTL5 in the regulation of translation.
    Keywords:  18S; ER-UPR; METTL5; S6K; decoding center; lifespan; m6A1832; rRNA modification; ribosome; translation initiation
    DOI:  https://doi.org/10.1016/j.celrep.2020.108544
  23. Antioxidants (Basel). 2020 Dec 22. pii: E1. [Epub ahead of print]10(1):
    Esencan E, Cozzolino M, Imamoglu G, Seli E.
      Mitochondrial unfolded protein response (UPRmt) is a highly conserved mechanism, which is activated upon cellular or metabolic stress and aims to help cells maintain homeostasis. CLPP (caseinolytic peptidase P) plays a crucial factor for UPRmt; it promotes the degradation of unfolded mitochondrial proteins. Global germline deletion of Clpp in mice results in female infertility and accelerated follicular depletion. Here, we asked whether CLPP is necessary for granulosa/cumulus cell function. Clppflox/flox mice were generated and crossbred with Cyp19a1-Cre mice to generate mice with granulosa/cumulus cell-specific Clpp deletion (Clpp-/-). Mature (8-week-old) Clpp-/- female mice (8-week-old) were compared to same age wild type (WT) mice. We found that mature Clpp-/- female mice were fertile and produced a similar number of pups per litter compared to WT. Folliculogenesis was not affected by the loss of CLPP in granulosa/cumulus cells as Clpp-/- and WT mice had a similar number of primordial, primary, secondary, early antral, and antral follicles. The number of germinal vesicles (GV) and MII oocytes collected from Clpp-/- and WT female mice were also similar. Our findings demonstrate that fertility in female mice is not affected by granulosa/cumulus cell-specific UPRmt disruption through CLPP deletion.
    Keywords:  mitochondrial UPR; mitochondrial stress; ovarian aging
    DOI:  https://doi.org/10.3390/antiox10010001
  24. Sci Adv. 2020 Dec;pii: eabd6078. [Epub ahead of print]6(51):
    Han XR, Sasaki N, Jackson SC, Wang P, Li Z, Smith MD, Xie L, Chen X, Zhang Y, Marzluff WF, Xiong Y.
      Evolutionarily conserved DCAF1 is a major substrate receptor for the DDB1-CUL4-ROC1 E3 ubiquitin ligase (CRL4) and controls cell proliferation and development. The molecular basis for these functions is unclear. We show here that DCAF1 loss in multiple tissues and organs selectively eliminates proliferating cells and causes perinatal lethality, thymic atrophy, and bone marrow defect. Inducible DCAF1 loss eliminates proliferating, but not quiescent, T cells and MEFs. We identify the ribosome assembly factor PWP1 as a substrate of the CRL4DCAF1 ligase. DCAF1 loss results in PWP1 accumulation, impairing rRNA processing and ribosome biogenesis. Knockdown or overexpression of PWP1 can rescue defects or cause similar defects as DCAF1 loss, respectively, in ribosome biogenesis. DCAF1 loss increases free RPL11, resulting in L11-MDM2 association and p53 activation. Cumulatively, these results reveal a critical function for DCAF1 in ribosome biogenesis and define a molecular basis of DCAF1 function in cell proliferation and development.
    DOI:  https://doi.org/10.1126/sciadv.abd6078
  25. Cell Biol Int. 2020 Dec 16.
    Xu W, Wang C, Hua J.
      The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes endoplasmic reticulum stress (ERS), which is characteristic of cells with high levels of secretory activity and is involved in a variety of diseases. In response to ERS, cells initiate an adaptive process named the unfolding protein response (UPR) to maintain intracellular homeostasis and survival. However, long term and unresolved ERS can also induce apoptosis. As the most conserved signaling branch of UPR, the IRE1-XBP1 pathway plays an important role in both physiological and pathological states, and its activity has a profound impact on disease progression and prognosis. Here, the latest research progress of IRE1-XBP1 pathway in cancer, metabolic diseases, and other diseases was briefly introduced, and the relationship between several diseases and this pathway was analyzed. Besides, the new understanding and prospect of IRE1-XBP1 pathway regulating male reproduction were reviewed.
    Keywords:  ER stress; IRE1α; XBP1; disease
    DOI:  https://doi.org/10.1002/cbin.11533
  26. Clin Immunol. 2020 Dec 25. pii: S1521-6616(20)30819-6. [Epub ahead of print]223 108659
    Li M, Xie Y, Zhao K, Chen K, Cao Y, Zhang J, Han M, Hu L, He R, Wang D, Li H.
      Endoplasmic reticulum (ER) stress results in the activation of the unfolded protein response (UPR), a process that is involved in the pathogenesis of many inflammatory diseases. However, the role of ER stress in chronic rhinosinusitis with nasal polyps (CRSwNP) has yet to be elucidated. In this study, we found that the protein expression levels of a range of ER stress regulators, including p-PERK, ATF4, ATF6 and XBP1s, were significantly increased in CRSwNP compared to controls. Importantly, the expression of ATF4 and XBP1s was positively correlated with heightened inflammation in CRSwNP. In human nasal epithelial cells, the ER stress inducer tunicamycin (TM) could potentiate Toll-like receptors (TLRs) induced proinflammatory cytokines production. Furthermore, we found that the silencing of XBP1, but not ATF4 or ATF6, abrogated the proinflammatory effect of TM. Mechanistically, ER stress did not affect the NF-κB, MAPK or IRF3 signaling pathways. However, the ER stress regulator XBP1s was able to bind directly to the promoter region of inflammatory genes to modulate gene transcription. Besides, the commensal bacteria Staphylococcus aureus and several inflammatory factors, such as IL4, IL13, IL17 and IFNγ, could induce ER stress in epithelial cells. Collectively, ER stress plays a crucial role in facilitating TLR-induced inflammation. Targeting XBP1 can inhibit the inflammatory response, thus offering a potential approach to treat CRSwNP.
    Keywords:  Chronic rhinosinusitis with nasal polyps; ER stress; Inflammation; Toll like receptors; XBP1
    DOI:  https://doi.org/10.1016/j.clim.2020.108659
  27. Cell Rep. 2020 Dec 22. pii: S2211-1247(20)31525-4. [Epub ahead of print]33(12): 108536
    Wojnacki J, Nola S, Bun P, Cholley B, Filippini F, Pressé MT, Lipecka J, Man Lam S, N'guyen J, Simon A, Ouslimani A, Shui G, Fader CM, Colombo MI, Guerrera IC, Galli T.
      VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth.
    Keywords:  autophagy, ER-phagy, secretion, neuron, development, SNARE, VAMP7, ATG5, RTN3, ATL3
    DOI:  https://doi.org/10.1016/j.celrep.2020.108536
  28. Biochem Biophys Res Commun. 2020 Dec 23. pii: S0006-291X(20)32231-2. [Epub ahead of print]536 45-51
    Rao V, Virupapuram V.
      Plants experience abiotic stresses throughout their life cycle and accordingly respond to tide over the unfavorable conditions. Plants adopt to drought stress through various molecular, biochemical, physiological and cellular processes. F-box protein subunit of the Skp1-Cullin-F-box (SCF) E3 ubiquitin ligases plays crucial role in imparting specificity for selective degradation of target proteins. Here we report the function of Arabidopsis F-box protein At1g08710 in drought stress adaptation. F-box protein is a constituent of SCF complex as it is shown interacting with ASK1 and Cullin 1. F-box protein localizes in both nucleus and membrane. F-box gene transcript is highly accumulated in root and altered in response to drought stress conditions. F-box protein interacts with a transcriptional co-activator protein ADA2b. F-box mutant plants growth is better under drought stress conditions compared to the wild type. Accumulation of H2O2 and malondialdehyde (MDA) content is reduced in mutant plants. Drought responsive genes RD29A, RD22, ABI3 expression is induced in F-box mutant plants. These results indicate F-box protein At1g08710 role in drought stress adaptation in Arabidopsis thaliana.
    Keywords:  Abscisic acid; Arabidopsis thaliana; Drought stress; F-box protein
    DOI:  https://doi.org/10.1016/j.bbrc.2020.12.054
  29. Cell Rep. 2020 Dec 29. pii: S2211-1247(20)31553-9. [Epub ahead of print]33(13): 108564
    Wrobel L, Siddiqi FH, Hill SM, Son SM, Karabiyik C, Kim H, Rubinsztein DC.
      The mechanistic target of rapamycin complex 2 (mTORC2) controls cell metabolism and survival in response to environmental inputs. Dysregulation of mTORC2 signaling has been linked to diverse human diseases, including cancer and metabolic disorders, highlighting the importance of a tightly controlled mTORC2. While mTORC2 assembly is a critical determinant of its activity, the factors regulating this event are not well understood, and it is unclear whether this process is regulated by growth factors. Here, we present data, from human cell lines and mice, describing a mechanism by which growth factors regulate ubiquitin-specific protease 9X (USP9X) deubiquitinase to stimulate mTORC2 assembly and activity. USP9X removes Lys63-linked ubiquitin from RICTOR to promote its interaction with mTOR, thereby facilitating mTORC2 signaling. As mTORC2 is central for cellular homeostasis, understanding the mechanisms regulating mTORC2 activation toward its downstream targets is vital for our understanding of physiological processes and for developing new therapeutic strategies in pathology.
    Keywords:  RICTOR; USP9X; growth factor signaling; mTORC2; mechanistic target of rapamycin complex 2; posttranslational modification; ubiquitin-specific protease 9X
    DOI:  https://doi.org/10.1016/j.celrep.2020.108564
  30. Nat Neurosci. 2020 Dec 23.
    Kalish BT, Kim E, Finander B, Duffy EE, Kim H, Gilman CK, Yim YS, Tong L, Kaufman RJ, Griffith EC, Choi GB, Greenberg ME, Huh JR.
      Maternal infection and inflammation during pregnancy are associated with neurodevelopmental disorders in offspring, but little is understood about the molecular mechanisms underlying this epidemiologic phenomenon. Here, we leveraged single-cell RNA sequencing to profile transcriptional changes in the mouse fetal brain in response to maternal immune activation (MIA) and identified perturbations in cellular pathways associated with mRNA translation, ribosome biogenesis and stress signaling. We found that MIA activates the integrated stress response (ISR) in male, but not female, MIA offspring in an interleukin-17a-dependent manner, which reduced global mRNA translation and altered nascent proteome synthesis. Moreover, blockade of ISR activation prevented the behavioral abnormalities as well as increased cortical neural activity in MIA male offspring. Our data suggest that sex-specific activation of the ISR leads to maternal inflammation-associated neurodevelopmental disorders.
    DOI:  https://doi.org/10.1038/s41593-020-00762-9
  31. Cell Microbiol. 2020 Dec 23.
    Friedrich A, Beare PA, Schulze-Luehrmann J, Cordsmeier A, Pazen T, Sonnewald S, Lührmann A.
      The obligate intracellular pathogen C. burnetii is the causative agent of the zoonosis Q fever. C. burnetii infection can have severe outcomes due to the development of chronic infection. To establish and maintain an infection, C. burnetii depends on a functional type IVB secretion system (T4BSS) and, thus, on the translocation of effector proteins into the host cell. Here, we showed that the C. burnetii T4BSS effector protein CaeB targets the conserved ER stress sensor IRE1 during ER stress in mammalian and plant cells. CaeB-induced upregulation of IRE1 RNase activity was essential for CaeB-mediated inhibition of ER stress-induced cell death. Our data reveal a novel role for CaeB in ER stress signaling modulation and demonstrate that CaeB is involved in pathogenicity in vivo. Furthermore, we provide evidence that C. burnetii infection leads to modulation of the ER stress sensors IRE1 and PERK, but not ATF6 during ER stress. While the upregulation of the RNase activity of IRE1 during ER stress depends on CaeB, modulation of PERK is CaeB independent, suggesting that C. burnetii encodes several factors influencing ER stress during infection. This article is protected by copyright. All rights reserved.
    Keywords:  Coxiella burnetii; ER stress; Nicotiana benthamiana; apoptosis; effector proteins; type IV secretion system
    DOI:  https://doi.org/10.1111/cmi.13305
  32. J Cell Biol. 2021 Feb 01. pii: e202009167. [Epub ahead of print]220(2):
    Barbosa P, Zhaunova L, Debilio S, Steccanella V, Kelly V, Ly T, Ohkura H.
      Meiosis creates genetic diversity by recombination and segregation of chromosomes. The synaptonemal complex assembles during meiotic prophase I and assists faithful exchanges between homologous chromosomes, but how its assembly/disassembly is regulated remains to be understood. Here, we report how two major posttranslational modifications, phosphorylation and ubiquitination, cooperate to promote synaptonemal complex assembly. We found that the ubiquitin ligase complex SCF is important for assembly and maintenance of the synaptonemal complex in Drosophila female meiosis. This function of SCF is mediated by two substrate-recognizing F-box proteins, Slmb/βTrcp and Fbxo42. SCF-Fbxo42 down-regulates the phosphatase subunit PP2A-B56, which is important for synaptonemal complex assembly and maintenance.
    DOI:  https://doi.org/10.1083/jcb.202009167
  33. J Cell Mol Med. 2020 Dec 25.
    Feng P, Li L, Dai J, Zhou L, Liu J, Zhao J, Li X, Ling N, Qiu S, Zhang L, Xie T, Chen Y, Donovan MJ, Peng T, Song J, Ye M.
      Ubiquitin-specific protease 11 (USP11) has been implicated in the regulation of DNA repair, apoptosis, signal transduction and cell cycle. It belongs to a USP subfamily of deubiquitinases. Although previous research has shown that USP11 overexpression is frequently found in melanoma and is correlated with a poor prognosis, the potential molecular mechanism of USP11 in melanoma remains indefinitive. Here, we report that USP11 and NONO colocalize and interact with each other in the nucleus of melanoma cells. As a result, the knockdown of USP11 decreases NONO levels. Whereas, overexpression of USP11 increases NONO levels in a dose-dependent manner. Furthermore, we reveal that USP11 protects NONO protein from proteasome-mediated degradation by removing poly-ubiquitin chains conjugated onto NONO. Functionally, USP11 mediated melanoma cell proliferation via the regulation of NONO levels because ablation of USP11 inhibits the proliferation which could be rescued by ectopic expression of NONO protein. Moreover, a significant positive correlation between USP11 and NONO concentrations was found in clinical melanoma samples. Collectively, these results demonstrate that USP11 is a new deubiquitinase of NONO and that the signalling axis of USP11-NONO is significantly involved in melanoma proliferation.
    Keywords:  NONO; USP11; deubiquitination; melanoma; proliferation
    DOI:  https://doi.org/10.1111/jcmm.16243
  34. EMBO J. 2020 Dec 28. e105120
    Morleo M, Brillante S, Formisano U, Ferrante L, Carbone F, Iaconis D, Palma A, Buonomo V, Maione AS, Grumati P, Settembre C, Franco B.
      Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.
    Keywords:  OFD1; autophagy receptor; polycystic kidney; selective autophagy
    DOI:  https://doi.org/10.15252/embj.2020105120
  35. FEBS Lett. 2020 Dec 23.
    Qin S, Ye L, Zheng Y, Gao J.
      Mutations in PINK1 (PTEN-induced putative kinase 1) are associated with autosomal recessive early-onset Parkinson's disease. Full-length PINK1 (PINK1-l) has been extensively studied in mitophagy, however, the functions of the short form of PINK1 (PINK1-s) remain poorly understood. Here, we report that PINK1-s is recruited to ribosome fractions after short-term inhibition of proteasomes. Expression of PINK1-s greatly inhibits protein synthesis even without proteasomal stress. Mechanistically, PINK1-s phosphorylates the translation elongation factor eEF1A1 during proteasome inhibition. Expression of the phosphorylation mimic mutation eEF1A1S396E rescues protein synthesis defects and cell viability caused by PINK1 knockout. These findings implicate an important role for PINK1-s in protecting cells against proteasome stress through inhibiting protein synthesis.
    Keywords:  PINK1; eEF1A1; phosphorylation; proteasome inhibition; protein synthesis; stress response
    DOI:  https://doi.org/10.1002/1873-3468.14030
  36. Cell Rep. 2020 Dec 29. pii: S2211-1247(20)31522-9. [Epub ahead of print]33(13): 108533
    van den Berk P, Lancini C, Company C, Serresi M, Sanchez-Bailon MP, Hulsman D, Pritchard C, Song JY, Schmitt MJ, Tanger E, Popp O, Mertins P, Huijbers IJ, Jacobs H, van Lohuizen M, Gargiulo G, Citterio E.
      Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens. We find that multiple DUBs may be individually required for hematopoiesis and identify ubiquitin-specific protease 15 (USP15) as essential for HSC maintenance in vitro and in transplantations and Usp15 knockout (KO) mice in vivo. USP15 is highly expressed in human hematopoietic tissues and leukemias. USP15 depletion in murine progenitors and leukemia cells impairs in vitro expansion and increases genotoxic stress. In leukemia cells, USP15 interacts with and stabilizes FUS (fused in sarcoma), a known DNA repair factor, directly linking USP15 to the DNA damage response (DDR). Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSCs and leukemia cells.
    Keywords:  DNA damage response; FUS; HSC; RNAi; USP15; deubiquitinase; deubiquitinating enzymes; fused in sarcoma; genome integrity; hematopoietic stem cell; in vivo shRNA screen; leukemia
    DOI:  https://doi.org/10.1016/j.celrep.2020.108533
  37. J Cell Sci. 2020 Dec 29. pii: jcs249136. [Epub ahead of print]133(24):
    Cremer T, Neefjes J, Berlin I.
      Calcium is the third most abundant metal on earth, and the fundaments of its homeostasis date back to pre-eukaryotic life forms. In higher organisms, Ca2+ serves as a cofactor for a wide array of (enzymatic) interactions in diverse cellular contexts and constitutes the most important signaling entity in excitable cells. To enable responsive behavior, cytosolic Ca2+ concentrations are kept low through sequestration into organellar stores, particularly the endoplasmic reticulum (ER), but also mitochondria and lysosomes. Specific triggers are then used to instigate a local release of Ca2+ on demand. Here, communication between organelles comes into play, which is accomplished through intimate yet dynamic contacts, termed membrane contact sites (MCSs). The field of MCS biology in relation to cellular Ca2+ homeostasis has exploded in recent years. Taking advantage of this new wealth of knowledge, in this Review, we invite the reader on a journey of Ca2+ flux through the ER and its associated MCSs. New mechanistic insights and technological advances inform the narrative on Ca2+ acquisition and mobilization at these sites of communication between organelles, and guide the discussion of their consequences for cellular physiology.
    Keywords:  Calcium; ER; Endosome; Membrane contact sites; Mitochondria
    DOI:  https://doi.org/10.1242/jcs.249136
  38. J Exp Med. 2021 Feb 01. pii: e20201217. [Epub ahead of print]218(2):
    Wang A, Ding L, Wu Z, Ding R, Teng XL, Wang F, Hu Z, Chen L, Yu X, Zou Q.
      Autophagy programs the metabolic and functional fitness of regulatory T (T reg) cells to establish immune tolerance, yet the mechanisms governing autophagy initiation in T reg cells remain unclear. Here, we show that the E3 ubiquitin ligase ZFP91 facilitates autophagy activation to sustain T reg cell metabolic programming and functional integrity. T reg cell-specific deletion of Zfp91 caused T reg cell dysfunction and exacerbated colonic inflammation and inflammation-driven colon carcinogenesis. TCR-triggered autophagy induction largely relied on T reg cell-derived ZFP91 to restrict hyperglycolysis, which is required for the maintenance of T reg cell homeostasis. Mechanistically, ZFP91 rapidly translocated from the nucleus to the cytoplasm in response to TCR stimulation and then mediated BECN1 ubiquitination to promote BECN1-PIK3C3 complex formation. Therefore, our results highlight a ZFP91-dependent mechanism promoting TCR-initiated autophagosome maturation to maintain T reg cell homeostasis and function.
    DOI:  https://doi.org/10.1084/jem.20201217
  39. Cells. 2020 12 10. pii: E2652. [Epub ahead of print]9(12):
    Frappaolo A, Karimpour-Ghahnavieh A, Sechi S, Giansanti MG.
      Glycosylation is the most common post-translational modification of proteins; it mediates their correct folding and stability, as well as their transport through the secretory transport. Changes in N- and O-linked glycans have been associated with multiple pathological conditions including congenital disorders of glycosylation, inflammatory diseases and cancer. Glycoprotein glycosylation at the Golgi involves the coordinated action of hundreds of glycosyltransferases and glycosidases, which are maintained at the correct location through retrograde vesicle trafficking between Golgi cisternae. In this review, we describe the molecular machinery involved in vesicle trafficking and tethering at the Golgi apparatus and the effects of mutations in the context of glycan biosynthesis and human diseases.
    Keywords:  GOLPH3; GORAB; Golgi; glycosylation; oligomeric golgi complex
    DOI:  https://doi.org/10.3390/cells9122652
  40. Mol Biol Cell. 2020 Dec 30. mbcE20100622
    Menon S, Goldfarb D, Ho CT, Cloer EW, Boyer NP, Hardie C, Bock AJ, Johnson EC, Anil J, Major MB, Gupton SL.
      TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified putative neuronal TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high priority candidates was validated, including Myo16, Coro1A, MAP1B, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized TIRF microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated the RNAi-based knockdown of the unconventional myosin Myo16 in cortical neurons altered growth cone filopodia density and axonal branching patterns in a TRIM9 and netrin-1 dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function.
    DOI:  https://doi.org/10.1091/mbc.E20-10-0622
  41. Sci Adv. 2020 Dec;pii: eabd0263. [Epub ahead of print]6(51):
    Sučec I, Wang Y, Dakhlaoui O, Weinhäupl K, Jores T, Costa D, Hessel A, Brennich M, Rapaport D, Lindorff-Larsen K, Bersch B, Schanda P.
      Chaperones are essential for assisting protein folding and for transferring poorly soluble proteins to their functional locations within cells. Hydrophobic interactions drive promiscuous chaperone-client binding, but our understanding of how additional interactions enable client specificity is sparse. Here, we decipher what determines binding of two chaperones (TIM8·13 and TIM9·10) to different integral membrane proteins, the all-transmembrane mitochondrial carrier Ggc1 and Tim23, which has an additional disordered hydrophilic domain. Combining NMR, SAXS, and molecular dynamics simulations, we determine the structures of Tim23/TIM8·13 and Tim23/TIM9·10 complexes. TIM8·13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the transmembrane part are weaker than in TIM9·10. Consequently, TIM9·10 outcompetes TIM8·13 in binding hydrophobic clients, while TIM8·13 is tuned to few clients with both hydrophilic and hydrophobic parts. Our study exemplifies how chaperones fine-tune the balance of promiscuity versus specificity.
    DOI:  https://doi.org/10.1126/sciadv.abd0263
  42. Cell Cycle. 2020 Dec 31. 1-15
    Chauhan AK, Li P, Sun Y, Wani G, Zhu Q, Wani AA.
      Mineralocorticoid and androgen receptor antagonist, spironolactone, was recently identified as an inhibitor of nucleotide excision repair (NER), acting via induction of proteolysis of TFIIH component Xeroderma Pigmentosum B protein (XPB). This activity provides a strong rationale for repurposing spironolactone for cancer therapy. Here, we report that the spironolactone-induced XPB proteolysis is mediated through ubiquitin-selective segregase, valosin-containing protein (VCP)/p97. We show that spironolactone induces a dose- and time-dependent degradation of XPB but not XPD, and that the XPB degradation is blocked by VCP/p97 inhibitors DBeQ, NMS-873, and neddylation inhibitor MLN4924. Moreover, the cellular treatment by VCP/p97 inhibitors leads to the accumulation of ubiquitin conjugates of XPB but not XPD. VCP/p97 knockdown by inducible shRNA does not affect XPB level but compromises the spironolactone-induced XPB degradation. Also, VCP/p97 interacts with XPB upon treatment of spironolactone and proteasome inhibitor MG132, while the VCP/p97 adaptor UBXD7 binds XPB and its ubiquitin conjugates. Additionally, ATP analog-mediated inhibition of Cdk7 significantly decelerates spironolactone-induced XPB degradation. Likewise, engaging TFIIH to NER by UV irradiation slows down spironolactone-induced XPB degradation. These results indicate that the spironolactone-induced XPB proteolysis requires VCP/p97 function and that XPB within holo-TFIIH rather than core-TFIIH is more vulnerable to spironolactone-induced proteolysis. Abbreviations NER: nucleotide excision repair; TFIIH: transcription factor II H; CAK: Cdk-activating kinase (CAK) complex; XPB: Xeroderma Pigmentosum type B; VCP/p97: valosin-containing protein/p97; Cdk7: cyclin-dependent kinase 7; NAE: NEDD8-activating enzyme; IP: immunoprecipitation.
    Keywords:  Cdk-activating kinase complex; Nucleotide excision repair; VCP/p97; cyclin-dependent kinase 7; neddylation; proteasome; spironolactone; transcription factor II H; xeroderma pigmentosum type B
    DOI:  https://doi.org/10.1080/15384101.2020.1860559
  43. FASEB J. 2021 Jan;35(1): e21265
    Li B, Lee C, Chuslip S, Lee D, Biouss G, Wu R, Koike Y, Miyake H, Ip W, Gonska T, Pierro A.
      Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases affecting premature infants. It has been shown that NEC is associated with disrupted intestinal barrier and dysregulated endoplasmic reticulum (ER)-stress response. It has also been shown that stem cells derived from amniotic fluid (AFSC) rescued intestinal injury in experimental NEC. Herein, we hypothesized that the beneficial effects of AFSC in the injured intestine are due to the restoration of intestinal barrier function. We evaluated intestinal barrier function using an ex vivo intestinal organoid model of NEC. We found that AFSC restored the expression and localization of tight junction proteins in intestinal organoids, and subsequently decreased epithelial permeability. AFSC rescued tight junction expression by inducing a protective ER stress response that prevents epithelial cell apoptosis in injured intestinal organoids. Finally, we validated these results in our experimental mouse model of NEC and confirmed that AFSC induced sustained ER stress and prevented intestinal apoptosis. This response led to the restoration of tight junction expression and localization, which subsequently reduced intestinal permeability in NEC pups. These findings confirm that intestinal barrier function is disrupted during NEC intestinal injury, and further demonstrate the disruption can be reversed by the administration of AFSC through the activation of the ER stress pathway. This study provides insight into the pathogenesis of NEC and highlights potential therapeutic targets for the treatment of NEC.
    Keywords:  amniotic fluid stem cells; endoplasmic reticulum stress; intestine permeability; necrotizing enterocolitis; tight junction
    DOI:  https://doi.org/10.1096/fj.202001426R
  44. Plant Cell Physiol. 2020 Dec 24. pii: pcaa157. [Epub ahead of print]
    Isono K, Tsukimoto R, Iuchi S, Shinozawa A, Yotsui I, Sakata Y, Taji T.
      Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days. To reveal the mechanisms underlying L-heat stress tolerance, we here used a forward genetic screening for sensitive to long-term heat (sloh) mutants and isolated sloh4. The mutant was hypersensitive to L- but not S-heat stress. The causal gene of sloh4 was identical to MIP3 encoding a member of the MAIGO2 (MAG2) tethering complex, which is composed of the MAG2, MIP1, MIP2, and MIP3 subunits and is localized at the endoplasmic reticulum (ER) membrane. Although sloh4/mip3 was hypersensitive to L-heat stress, the sensitivity of the mag2-3 and mip1-1 mutants was similar to that of the wild type. Under L-heat stress, the ER stress and the following unfolded protein response (UPR) were more pronounced in sloh4 than in the wild type. Transcript levels of bZIP60-regulated UPR genes were strongly increased in sloh4 under L-heat stress. Two processes known to be mediated by INOSITOL REQUIRING ENZYME1 (IRE1)-accumulation of the spliced bZIP60 transcript and a decrease in the transcript levels of PR4 and PRX34, encoding secretory proteins-were observed in sloh4 in response to L-heat stress. These findings suggest that misfolded proteins generated in sloh4 under L-heat stress may be recognized by IRE1 but not bZIP28, resulting in initiation of the UPR via activated bZIP60. Therefore, it would be possible that only MIP3 in MAG2 complex has an additional function in L-heat tolerance, which is not related to the ER-Golgi vesicle tethering.
    Keywords:   Arabidopsis thaliana ; ER stress; UPR; heat; long-term heat; tolerance
    DOI:  https://doi.org/10.1093/pcp/pcaa157
  45. Science. 2021 Jan 01. 371(6524): 57-64
    Bertolini M, Fenzl K, Kats I, Wruck F, Tippmann F, Schmitt J, Auburger JJ, Tans S, Bukau B, Kramer G.
      Accurate assembly of newly synthesized proteins into functional oligomers is crucial for cell activity. In this study, we investigated whether direct interaction of two nascent proteins, emerging from nearby ribosomes (co-co assembly), constitutes a general mechanism for oligomer formation. We used proteome-wide screening to detect nascent chain-connected ribosome pairs and identified hundreds of homomer subunits that co-co assemble in human cells. Interactions are mediated by five major domain classes, among which N-terminal coiled coils are the most prevalent. We were able to reconstitute co-co assembly of nuclear lamin in Escherichia coli, demonstrating that dimer formation is independent of dedicated assembly machineries. Co-co assembly may thus represent an efficient way to limit protein aggregation risks posed by diffusion-driven assembly routes and ensure isoform-specific homomer formation.
    DOI:  https://doi.org/10.1126/science.abc7151
  46. Mol Cancer Res. 2020 Dec 30. pii: molcanres.0579.2020. [Epub ahead of print]
    Liu X, Lu Y, Chen Z, Liu X, Hu W, Zheng L, Chen Y, Kurie JM, Shi M, Mustachio LM, Andresson T, Fox S, Roszik J, Kawakami M, Freemantle S, Dmitrovsky E.
      Ubiquitin specific protease18 (USP18), previously known as UBP43, is the Interferon-Stimulated Gene 15 (ISG15) deconjugase. USP18 removes ISG15 from substrate proteins. This study reports that USP18 null mice (versus wild-type mice) exhibited lower lipolysis rates, altered fat to body weight ratios and cold sensitivity. USP18 is a regulator of lipid and fatty acid metabolism. Prior work established that USP18 promotes lung tumorigenesis. We sought to learn if this occurs through altered lipid and fatty acid metabolism. Loss of USP18 repressed adipose triglyceride lipase (ATGL) expression; gain of USP18 expression upregulated ATGL in lung cancer cells. The E1-like ubiquitin activating enzyme (UBE1L) promoted ISG15-conjugation of ATGL and destabilization. Immunoprecipitation assays confirmed that ISG15 covalently conjugates to ATGL. Protein expression of thermogenic regulators was examined in brown fat of USP18 null versus wild-type mice. Uncoupling Protein 1 (UCP1) was repressed in USP18 null fat. Gain of USP18 expression augmented UCP1 protein via reduced ubiquitination. Gain of UCP1 expression in lung cancer cell lines enhanced cellular proliferation. UCP1 knock-down inhibited proliferation. Beta-hydroxybutyrate colorimetric assays performed after gain of UCP1 expression revealed increased cellular fatty acid beta-oxidation, augmenting fatty acid beta-oxidation in seahorse assays. Combined USP18, ATGL and UCP1 profiles were interrogated in The Cancer Genome Atlas (TCGA). Intriguingly, lung cancers with increased USP18, ATGL and UCP1 expression had an unfavorable survival. These findings reveal that USP18 is a pharmacologic target that controls fatty acid metabolism. Implications: USP18 is an antineoplastic target that affects lung cancer fatty acid metabolism.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0579
  47. J Biol Chem. 2020 Dec 31. pii: jbc.RA120.016116. [Epub ahead of print]
    Seepersaud R, Anderson AC, Bensing BA, Choudhury BP, Clarke AJ, Sullam PM.
      The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec (aSec) system. While all aSec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin GspB. Since these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues, and that O-acetylation prevented Glc deposition. Whereas streptococci expressing non-acetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to wild-type levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, since O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.
    Keywords:  Streptococcus; acetylation; adhesin; glycoprotein biosynthesis; glycosylation
    DOI:  https://doi.org/10.1074/jbc.RA120.016116
  48. J Virol. 2020 Dec 23. pii: JVI.02351-20. [Epub ahead of print]
    Wang S, Hou P, Pan W, He W, He DC, Wang H, He H.
      DNA damage inducible transcript 3 (DDIT3) plays important roles in ER stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent OTU deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation.IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 was positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.
    DOI:  https://doi.org/10.1128/JVI.02351-20
  49. J Neural Transm (Vienna). 2021 Jan 01.
    Gonzalez-Latapi P, Marotta N, Mencacci NE.
      Dystonia is a clinically, genetically, and biologically heterogeneous hyperkinetic movement disorder caused by the dysfunctional activity of neural circuits involved in motor control. Our understanding of the molecular mechanisms underlying dystonia pathogenesis has tremendously grown thanks to the accelerated discovery of genes associated with monogenic dystonias (DYT-genes). Genetic discoveries, together with the development of a growing number of cellular and animal models of genetic defects responsible for dystonia, are allowing the identification of several areas of functional convergence among the protein products of multiple DYT-genes. Furthermore, unexpected functional links are being discovered in the downstream pathogenic molecular mechanisms of DYT-genes that were thought to be unrelated based on their primary molecular functions. Examples of these advances are the recognition that multiple DYT-genes are involved in (1) endoplasmic reticulum function and regulation of the integrated stress response (ISR) through Eukaryotic initiation factor 2 alpha signaling; (2) gene transcription modulation during neurodevelopment; (3) pre-and post-synaptic nigrostriatal dopaminergic signaling; and (4) presynaptic neurotransmitter vesicle release. More recently, genetic defects in the endo-lysosomal and autophagy pathways have also been implicated in the molecular pathophysiology of dystonia, suggesting the existence of mechanistic overlap with other movement disorders, such as Parkinson's disease. Importantly, the recognition that multiple DYT-genes coalesce in shared biological pathways is a crucial advance in our understanding of dystonias and will aid in the development of more effective therapeutic strategies by targeting these convergent molecular pathways.
    Keywords:  Cellular stress response; Dopamine signaling; Dystonia; Gene transcription; Genetics; Molecular pathways; Synaptic transmission
    DOI:  https://doi.org/10.1007/s00702-020-02290-z
  50. STAR Protoc. 2020 Dec 18. 1(3): 100155
    Liu J, Xue Z, Vann KR, Shi X, Kutateladze TG.
      Since its discovery, several ligands of the ZZ domain have been identified; however, molecular and structural information underlying binding of these ligands remains limited. Here, we describe a protocol for biochemical and structural analysis of the ZZ domain of human E3 ubiquitin ligase HERC2 (HERC2ZZ) and its interaction with its ligands: the N-terminal tails of histone H3 and SUMO1. This methodology could be applied for characterization of binding activities of other histone readers. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).
    DOI:  https://doi.org/10.1016/j.xpro.2020.100155
  51. Sci Adv. 2020 Dec;pii: eabd7272. [Epub ahead of print]6(51):
    Yang B, Maddison LA, Zaborska KE, Dai C, Yin L, Tang Z, Zang L, Jacobson DA, Powers AC, Chen W.
      Islet inflammation is an important etiopathology of type 2 diabetes; however, the underlying mechanisms are not well defined. Using complementary experimental models, we discovered RIPK3-dependent IL1B induction in β cells as an instigator of islet inflammation. In cultured β cells, ER stress activated RIPK3, leading to NF-kB-mediated proinflammatory gene expression. In a zebrafish muscle insulin resistance model, overnutrition caused islet inflammation, β cell dysfunction, and loss in an ER stress-, ripk3-, and il1b-dependent manner. In mouse islets, high-fat diet triggered the IL1B expression in β cells before macrophage recruitment in vivo, and RIPK3 inhibition suppressed palmitate-induced β cell dysfunction and Il1b expression in vitro. Furthermore, in human islets grafted in hyperglycemic mice, a marked increase in ER stress, RIPK3, and NF-kB activation in β cells were accompanied with murine macrophage infiltration. Thus, RIPK3-mediated induction of proinflammatory mediators is a conserved, previously unrecognized β cell response to metabolic stress and a mediator of the ensuing islet inflammation.
    DOI:  https://doi.org/10.1126/sciadv.abd7272
  52. PLoS Pathog. 2020 Dec 22. 16(12): e1009134
    Stokes S, Almire F, Tatham MH, McFarlane S, Mertens P, Pondeville E, Boutell C.
      Mosquitoes are responsible for the transmission of many clinically important arboviruses that cause significant levels of annual mortality and socioeconomic health burden worldwide. Deciphering the mechanisms by which mosquitoes modulate arbovirus infection is crucial to understand how viral-host interactions promote vector transmission and human disease. SUMOylation is a post-translational modification that leads to the covalent attachment of the Small Ubiquitin-like MOdifier (SUMO) protein to host factors, which in turn can modulate their stability, interaction networks, sub-cellular localisation, and biochemical function. While the SUMOylation pathway is known to play a key role in the regulation of host immune defences to virus infection in humans, the importance of this pathway during arbovirus infection in mosquito vectors, such as Aedes aegypti (Ae. aegypti), remains unknown. Here we characterise the sequence, structure, biochemical properties, and tissue-specific expression profiles of component proteins of the Ae. aegypti SUMOylation pathway. We demonstrate significant biochemical differences between Ae. aegypti and Homo sapiens SUMOylation pathways and identify cell-type specific patterns of SUMO expression in Ae. aegypti tissues known to support arbovirus replication. Importantly, depletion of core SUMOylation effector proteins (SUMO, Ubc9 and PIAS) in Ae. aegypti cells led to enhanced levels of arbovirus replication from three different families; Zika (Flaviviridae), Semliki Forest (Togaviridae), and Bunyamwera (Bunyaviridae) viruses. Our findings identify an important role for mosquito SUMOylation in the cellular restriction of arboviruses that may directly influence vector competence and transmission of clinically important arboviruses.
    DOI:  https://doi.org/10.1371/journal.ppat.1009134
  53. Trends Cancer. 2020 Dec 19. pii: S2405-8033(20)30310-1. [Epub ahead of print]
    Kroonen JS, Vertegaal ACO.
      The small ubiquitin-like modifier (SUMO) signaling cascade is critical for gene expression, genome integrity, and cell cycle progression. In this review, we discuss the important role SUMO may play in cancer and how to target SUMO signaling. Recently developed small molecule inhibitors enable therapeutic targeting of the SUMOylation pathway. Blocking SUMOylation not only leads to reduced cancer cell proliferation but also to an increased antitumor immune response by stimulating interferon (IFN) signaling, indicating that SUMOylation inhibitors have a dual mode of action that can be employed in the fight against cancer. The search for tumor types that can be treated with SUMOylation inhibitors is ongoing. Employing SUMO conjugation inhibitory drugs in the years to come has potential as a new therapeutic strategy.
    Keywords:  SUMO; cancer; cell cycle; inhibitor; mitosis; ubiquitin
    DOI:  https://doi.org/10.1016/j.trecan.2020.11.009
  54. Front Pharmacol. 2020 ;11 582310
    Vela JM.
      Outbreaks of emerging infections, such as COVID-19 pandemic especially, confront health professionals with the unique challenge of treating patients. With no time to discover new drugs, repurposing of approved drugs or in clinical development is likely the only solution. Replication of coronaviruses (CoVs) occurs in a modified membranous compartment derived from the endoplasmic reticulum (ER), causes host cell ER stress and activates pathways to facilitate adaptation of the host cell machinery to viral needs. Accordingly, modulation of ER remodeling and ER stress response might be pivotal in elucidating CoV-host interactions and provide a rationale for new therapeutic, host-based antiviral approaches. The sigma-1 receptor (Sig-1R) is a ligand-operated, ER membrane-bound chaperone that acts as an upstream modulator of ER stress and thus a candidate host protein for host-based repurposing approaches to treat COVID-19 patients. Sig-1R ligands are frequently identified in in vitro drug repurposing screens aiming to identify antiviral compounds against CoVs, including severe acute respiratory syndrome CoV-2 (SARS-CoV-2). Sig-1R regulates key mechanisms of the adaptive host cell stress response and takes part in early steps of viral replication. It is enriched in lipid rafts and detergent-resistant ER membranes, where it colocalizes with viral replicase proteins. Indeed, the non-structural SARS-CoV-2 protein Nsp6 interacts with Sig-1R. The activity of Sig-1R ligands against COVID-19 remains to be specifically assessed in clinical trials. This review provides a rationale for targeting Sig-1R as a host-based drug repurposing approach to treat COVID-19 patients. Evidence gained using Sig-1R ligands in unbiased in vitro antiviral drug screens and the potential mechanisms underlying the modulatory effect of Sig-1R on the host cell response are discussed. Targeting Sig-1R is not expected to reduce dramatically established viral replication, but it might interfere with early steps of virus-induced host cell reprogramming, aid to slow down the course of infection, prevent the aggravation of the disease and/or allow a time window to mature a protective immune response. Sig-1R-based medicines could provide benefit not only as early intervention, preventive but also as adjuvant therapy.
    Keywords:  COVID-19; ER stress; SARS-CoV-2; anti-viral; drug repurposing; repurposed drugs; sigma-1 receptor; viral replication
    DOI:  https://doi.org/10.3389/fphar.2020.582310
  55. Proc Natl Acad Sci U S A. 2021 Jan 12. pii: e2024149118. [Epub ahead of print]118(2):
    Xiao X, Kim Y, Romartinez-Alonso B, Sirvydis K, Ory DS, Schwabe JWR, Jung ME, Tontonoz P.
      The Aster proteins (encoded by the Gramd1a-c genes) contain a ligand-binding fold structurally similar to a START domain and mediate nonvesicular plasma membrane (PM) to endoplasmic reticulum (ER) cholesterol transport. In an effort to develop small molecule modulators of Asters, we identified 20α-hydroxycholesterol (HC) and U18666A as lead compounds. Unfortunately, both 20α-HC and U18666A target other sterol homeostatic proteins, limiting their utility. 20α-HC inhibits sterol regulatory element-binding protein 2 (SREBP2) processing, and U18666A is an inhibitor of the vesicular trafficking protein Niemann-Pick C1 (NPC1). To develop potent and selective Aster inhibitors, we synthesized a series of compounds by modifying 20α-HC and U18666A. Among these, AI (Aster inhibitor)-1l, which has a longer side chain than 20α-HC, selectively bound to Aster-C. The crystal structure of Aster-C in complex with AI-1l suggests that sequence and flexibility differences in the loop that gates the binding cavity may account for the ligand specificity for Aster C. We further identified the U18666A analog AI-3d as a potent inhibitor of all three Aster proteins. AI-3d blocks the ability of Asters to bind and transfer cholesterol in vitro and in cells. Importantly, AI-3d also inhibits the movement of low-density lipoprotein (LDL) cholesterol to the ER, although AI-3d does not block NPC1. This finding positions the nonvesicular Aster pathway downstream of NPC1-dependent vesicular transport in the movement of LDL cholesterol to the ER. Selective Aster inhibitors represent useful chemical tools to distinguish vesicular and nonvesicular sterol transport mechanisms in mammalian cells.
    Keywords:  cholesterol; lipid metabolism; lipid transport
    DOI:  https://doi.org/10.1073/pnas.2024149118
  56. PLoS Genet. 2020 Dec;16(12): e1009252
    Srinivasan R, Walvekar AS, Rashida Z, Seshasayee A, Laxman S.
      Growth and starvation are considered opposite ends of a spectrum. To sustain growth, cells use coordinated gene expression programs and manage biomolecule supply in order to match the demands of metabolism and translation. Global growth programs complement increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis. How these resources are collectively managed is a fundamental question. The role of the Gcn4/ATF4 transcription factor has been best studied in contexts where cells encounter amino acid starvation. However, high Gcn4 activity has been observed in contexts of rapid cell proliferation, and the roles of Gcn4 in such growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum that maintains metabolic supply in order to sustain translation outputs. By integrating matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lysine and arginine enriched transcripts, which include genes encoding the translation machinery. The Gcn4 dependent lysine and arginine supply thereby maintains the synthesis of the translation machinery. This is required to maintain translation capacity. Gcn4 consequently enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controlled using the same Gcn4 transcriptional outputs that function in distinct contexts.
    DOI:  https://doi.org/10.1371/journal.pgen.1009252
  57. Angew Chem Int Ed Engl. 2020 Dec 25.
    Neuditschko B, Legin AA, Baier D, Schintlmeister A, Reipert S, Wagner M, Keppler BK, Berger W, Meier-Menches SM, Gerner C.
      The ruthenium-based anticancer agent BOLD-100/KP1339 has shown promising results in several in vitro and in vivo tumour models, as well as in early clinical trials. However, its mode of action remains to be fully elucidated. Recent evidence identified ER stress-induction and concomitant down-modulation of HSPA5 (GRP78) as key drug effects. By exploiting the naturally formed adduct between BOLD-100 and human serum albumin as an immobilization strategy, we were able to perform target profiling experiments that revealed the ribosomal proteins RPL10, RPL24 and the transcription factor GTF2I as potential interactors of this ruthenium(III) anticancer agent. Integrating these findings with proteomic profiling and transcriptomic experiments supported ribosomal disturbance and concomitant induction of endoplasmic reticulum (ER) stress. The formation of polyribosomes and ER swelling of treated cancer cells revealed by TEM validated this finding. Thus, the direct interaction of BOLD-100 with ribosomal proteins seems to accompany ER stress-induction and modulation of GRP78 in cancer cells.
    Keywords:  Bioinorganic Chemistry; Metals in Medicine; Ruthenium; multi-omics; ribosome
    DOI:  https://doi.org/10.1002/anie.202015962
  58. STAR Protoc. 2020 Dec 18. 1(3): 100217
    Amen T, Kaganovich D.
      Photoconversion enables real-time labeling of protein sub-populations inside living cells, which can then be tracked with submicrometer resolution. Here, we detail the protocol of comparing protein dynamics inside membraneless organelles in live HEK293T cells using a CRISPR-Cas9 PABPC1-Dendra2 marker of stress granules. Measuring internal dynamics of membraneless organelles provides insight into their functional state, physical properties, and composition. Photoconversion has the advantage over other imaging techniques in that it is less phototoxic and allows for dual color tracking of proteins. For complete details on the use and execution of this protocol, please refer to Amen and Kaganovich (2020).
    Keywords:  CRISPR; Cell Biology; Microscopy; Molecular/Chemical Probes
    DOI:  https://doi.org/10.1016/j.xpro.2020.100217
  59. Cancer Res. 2020 Dec 22. pii: canres.1777.2020. [Epub ahead of print]
    Bedekovics T, Hussain S, Zhang Y, Ali A, Jeon YJ, Galardy PJ.
      Deubiquitinating enzymes are increasingly recognized to play important roles in cancer, with many acting as oncogenes or tumor suppressors. In this study, we employed a bioinformatics approach to screen for enzymes from this family involved in cancer and found USP24 as a potent predictor of poor outcomes in neuroblastoma, an aggressive childhood cancer. USP24 resides in a region commonly deleted in neuroblastoma yet was independently associated with poor outcomes in this disease. Deletion of Usp24 in a murine model resulted in degradation of collapsin response mediator protein 2 (CRMP2), a regulator of axon growth, guidance, and neuronal polarity. Cells lacking USP24 had significant increases in spindle defects, chromosome missegregation, and aneuploidy; phenotypes that were rescued by the restoration of CRMP2. USP24 prevented aneuploidy by maintaining spindle-associated CRMP2, which is required for mitotic accuracy. Our findings further indicate that USP24 is a tumor suppressor that may play an important role in the pathogenesis of neuroblastoma.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-1777
  60. Matrix Biol. 2020 Dec 26. pii: S0945-053X(20)30119-0. [Epub ahead of print]
    Castagnaro S, Gambarotto L, Cescon M, Bonaldo P.
      Autophagy is a very versatile process through which the cell degrades damaged long-lived proteins, entire organelles, or pathogens, by engulfing them in characteristic double-membrane vesicles and conveying the cargo to lysosomes. It is a dynamic pathway tunable at multiple levels and responsive to nutrient and stress stimuli, also coming from the extracellular microenvironment and its remodeling. In the extracellular matrix, collagen type VI forms a distinctive set of beaded microfilaments that assemble into an intricate and multimodular meshwork of tightly linked proteins and surface receptors. When missing or defective, collagen VI triggers a series of pathological events in skeletal muscle and other tissues, with a remarkable impact on key cell processes, such as apoptosis and autophagy. In this review, we discuss the current knowledge about collagen VI regulation of autophagy in the different experimental models and human pathologies where it was studied, and provide some hints for future directions aimed at the fine dissection of this intriguing relationship, as well as its prospective translational impact for disease and therapy.
    Keywords:  Animal models; Autophagy; Collagen VI; Muscular dystrophies; Prospective therapies; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.matbio.2020.12.004