bims-proteo 2021-06-06 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2021–06–06
38 papers selected by
Eric Chevet, INSERM

TorsinA folding and N-linked glycosylation are sensitive to redox homeostasis.
Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.
Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis.
The Function of KDEL Receptors as UPR Genes in Disease.
IRE1α RIDD activity induced under ER stress drives neuronal death by the degradation of 14-3-3 θ mRNA in cortical neurons during glucose deprivation.
The deubiquitinating enzyme USP7 regulates the transcription factor Nrf1 by modulating its stability in response to toxic metal exposure.
Therapeutic Targeting of Protein Disulfide Isomerase PDIA1 in Multiple Myeloma.
In Vitro Analysis of E3 Ubiquitin Ligase Function.
HSPB1 influences mitochondrial respiration in ER-stressed beta cells.
Genome-wide imaging screen uncovers molecular determinants of arsenite-induced protein aggregation and toxicity.
The nonreceptor tyrosine kinase SRMS inhibits autophagy and promotes tumor growth by phosphorylating the scaffolding protein FKBP51.
The Carcinogen Cadmium Activates Lysine 63 (K63)-Linked Ubiquitin-Dependent Signaling and Inhibits Selective Autophagy.
The interplay between SUMOylation and phosphorylation of PKCδ facilitates oxidative stress-induced apoptosis.
ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs.
Alterations of the Platelet Proteome in Lung Cancer: Accelerated F13A1 and ER Processing as New Actors in Hypercoagulability.
UFL1 regulates milk protein and fat synthesis-related gene expression of bovine mammary epithelial cells probably via the mTOR signaling pathway.
ERO1-PDI redox signaling in health and disease.
Ubiquitin-Specific Protease 3 Deubiquitinates and Stabilizes Oct4 Protein in Human Embryonic Stem Cells.
Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex.
A proximity-dependent biotinylation map of a human cell.
MED12 interacts with the heat shock transcription factor HSF1 and recruits CDK8 to promote the heat shock response in mammalian cells.
Ubiquitination and Deubiquitination in Oral Disease.
Calcium-dependent and -independent lipid transfer mediated by tricalbins in yeast.
The parkinsonism-associated protein FBXO7 cooperates with the BAG6 complex in proteasome function and controls the subcellular localization of the complex.
SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A.
The GID ubiquitin ligase complex just reached the next level of complexity.
Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase.
Modification of small ubiquitin-related modifier 2 (SUMO2) by phosphoubiquitin in HEK293T cells.
Selective autophagy as the basis of autophagy-based degraders.
Origins and evolving functionalities of tRNA-derived small RNAs.
P300/HDAC1 regulates the acetylation/deacetylation and autophagic activities of LC3/Atg8-PE ubiquitin-like system.
Short and long sleeping mutants reveal links between sleep and macroautophagy.
OTULIN in NF-κB signaling, cell death, and disease.
Cryptic genetic variation in a heat shock protein modifies the outcome of a mutation affecting epidermal stem cell development in C. elegans.
Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.
To Ubiquitinate or Not to Ubiquitinate: TRIM17 in Cell Life and Death.
Variants in USP48 encoding ubiquitin hydrolase are associated with autosomal dominant non-syndromic hereditary hearing loss.
UBE4B, a microRNA-9 target gene, promotes autophagy-mediated Tau degradation.

Biochim Biophys Acta Mol Cell Res. 2021 May 29. pii: S0167-4889(21)00127-0. [Epub ahead of print] 119073

TorsinA folding and N-linked glycosylation are sensitive to redox homeostasis.

Jonas Honer, Katie M Niemeyer, Christian Fercher, Ana L Diez Tissera, Noushin Jaberolansar, Yohaann M A Jafrani, Chun Zhou, Julio J Caramelo, Annette M Shewan, Benjamin L Schulz, Jeffrey L Brodsky, Lucía F Zacchi.

  The Endoplasmic Reticulum (ER) is responsible for the folding and post-translational modification of secretory proteins, as well as for triaging misfolded proteins. During folding, there is a complex yet only partially understood interplay between disulfide bond formation, which is an enzyme catalyzed event in the oxidizing environment of the ER, along with other post-translational modifications (PTMs) and chaperone-supported protein folding. Here, we used the glycoprotein torsinA as a model substrate to explore the impact of ER redox homeostasis on PTMs and protein biogenesis. TorsinA is a AAA+ ATPase with unusual oligomeric properties and controversial functions. The deletion of a C-terminal glutamic acid residue (∆E) is associated with the development of Early-Onset Torsion Dystonia, a severe movement disorder. TorsinA differs from other AAA+ ATPases since it is an ER resident, and as a result of its entry into the ER torsinA contains two N-linked glycans and at least one disulfide bond. The role of these PTMs on torsinA biogenesis and function and the identity of the enzymes that catalyze them are poorly defined. Using a yeast torsinA expression system, we demonstrate that a specific protein disulfide isomerase, Pdi1, affects the folding and N-linked glycosylation of torsinA and torsinA∆E in a redox-dependent manner, suggesting that the acquisition of early torsinA folding intermediates is sensitive to perturbed interactions between Cys residues and the quality control machinery. We also highlight the role of specific Cys residues during torsinA biogenesis and demonstrate that torsinA∆E is more sensitive than torsinA when these Cys residues are mutated.

Keywords:  Dystonia; N-linked glycosylation; Post-translational modifications; Protein disulfide isomerase; Protein folding; torsinA

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119073
Int J Mol Sci. 2021 May 30. pii: 5859. [Epub ahead of print]22(11):

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

Ssu-Ju Fu, Meng-Chun Hu, Cheng-Tsung Hsiao, An-Ting Cheng, Tsung-Yu Chen, Chung-Jiuan Jeng, Chih-Yung Tang.

  The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Keywords:  17-AAG; channelopathy; chaperone; co-chaperone; protein quality control; proteostasis

DOI:  https://doi.org/10.3390/ijms22115859
Sci Rep. 2021 Jun 02. 11(1): 11562

Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis.

Peristera Roboti, Sarah O'Keefe, Kwabena B Duah, Wei Q Shi, Stephen High.

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

DOI: https://doi.org/10.1038/s41598-021-91107-4
Int J Mol Sci. 2021 May 21. pii: 5436. [Epub ahead of print]22(11):

The Function of KDEL Receptors as UPR Genes in Disease.

Emily S Wires, Kathleen A Trychta, Lacey M Kennedy, Brandon K Harvey.

  The KDEL receptor retrieval pathway is essential for maintaining resident proteins in the endoplasmic reticulum (ER) lumen. ER resident proteins serve a variety of functions, including protein folding and maturation. Perturbations to the lumenal ER microenvironment, such as calcium depletion, can cause protein misfolding and activation of the unfolded protein response (UPR). Additionally, ER resident proteins are secreted from the cell by overwhelming the KDEL receptor retrieval pathway. Recent data show that KDEL receptors are also activated during the UPR through the IRE1/XBP1 signaling pathway as an adaptive response to cellular stress set forth to reduce the loss of ER resident proteins. This review will discuss the emerging connection between UPR activation and KDEL receptors as it pertains to ER proteostasis and disease states.

Keywords:  ER resident proteins; KDEL receptor; disease; endoplasmic reticulum; exodosis; unfolded protein response

DOI:  https://doi.org/10.3390/ijms22115436
Cell Death Discov. 2021 Jun 03. 7(1): 131

IRE1α RIDD activity induced under ER stress drives neuronal death by the degradation of 14-3-3 θ mRNA in cortical neurons during glucose deprivation.

Juan Carlos Gómora-García, Cristian Gerónimo-Olvera, Xochitl Pérez-Martínez, Lourdes Massieu.

Altered protein homeostasis is associated with neurodegenerative diseases and acute brain injury induced under energy depletion conditions such as ischemia. The accumulation of damaged or unfolded proteins triggers the unfolded protein response (UPR), which can act as a homeostatic response or lead to cell death. However, the factors involved in turning and adaptive response into a cell death mechanism are still not well understood. Several mechanisms leading to brain injury induced by severe hypoglycemia have been described but the contribution of the UPR has been poorly studied. Cell responses triggered during both the hypoglycemia and the glucose reinfusion periods can contribute to neuronal death. Therefore, we have investigated the activation dynamics of the PERK and the IRE1α branches of the UPR and their contribution to neuronal death in a model of glucose deprivation (GD) and glucose reintroduction (GR) in cortical neurons. Results show a rapid activation of the PERK/p-eIF2α/ATF4 pathway leading to protein synthesis inhibition during GD, which contributes to neuronal adaptation, however, sustained blockade of protein synthesis during GR promotes neuronal death. On the other hand, IRE1α activation occurs early during GD due to its interaction with BAK/BAX, while ASK1 is recruited to IRE1α activation complex during GR promoting the nuclear translocation of JNK and the upregulation of Chop. Most importantly, results show that IRE1α RNase activity towards its splicing target Xbp1 mRNA occurs late after GR, precluding a homeostatic role. Instead, IRE1α activity during GR drives neuronal death by positively regulating ASK1/JNK activity through the degradation of 14-3-3 θ mRNA, a negative regulator of ASK and an adaptor protein highly expressed in brain, implicated in neuroprotection. Collectively, results describe a novel regulatory mechanism of cell death in neurons, triggered by the downregulation of 14-3-3 θ mRNA induced by the IRE1α branch of the UPR.

DOI: https://doi.org/10.1038/s41420-021-00518-9
J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00521-4. [Epub ahead of print] 100732

The deubiquitinating enzyme USP7 regulates the transcription factor Nrf1 by modulating its stability in response to toxic metal exposure.

John Jw Han, Daniel V Ho, Hyun M Kim, Jun Y Lee, Yerin S Jeon, Jefferson Y Chan.

  The Nuclear factor E2-related factor 1 (Nrf1) transcription factor performs a critical role in regulating cellular homeostasis as part of the cellular stress response, and drives the expression of antioxidants and detoxification enzymes among many other functions. Ubiquitination plays an important role in controlling the abundance and thus nuclear accumulation of Nrf1 proteins, but the regulatory enzymes that act on Nrf1 are not fully defined. Here, we identified ubiquitin specific protease 7 (USP7), a deubiquitinating enzyme, as a novel regulator of Nrf1 activity. We found that USP7 interacts with Nrf1a and TCF11-the two long protein isoforms of Nrf1. Expression of wild type USP7, but not its catalytically defective mutant, resulted in decreased ubiquitination of TCF11 and Nrf1a, leading to their increased stability, and increased transactivation of reporter gene expression by TCF11 and Nrf1a. In contrast, knockdown or pharmacologic inhibition of USP7 dramatically increased ubiquitination of TCF11 and Nrf1a, and reduction of their steady state levels. Loss of USP7 function attenuated the induction of Nrf1 protein expression in response to treatment with arsenic and other toxic metals, and inhibition of USP7 activity significantly sensitized cells to arsenic treatment. Collectively, these findings suggest that USP7 may act to modulate abundance of Nrf1 protein to induce gene expression in response to toxic metal exposure.

Keywords:  cellular stress; deubiquitinase; protein stability; toxic metals; transcription factor

DOI:  https://doi.org/10.1016/j.jbc.2021.100732
Cancers (Basel). 2021 May 28. pii: 2649. [Epub ahead of print]13(11):

Therapeutic Targeting of Protein Disulfide Isomerase PDIA1 in Multiple Myeloma.

Metis Hasipek, Dale Grabowski, Yihong Guan, Raghunandan Reddy Alugubelli, Anand D Tiwari, Xiaorong Gu, Gabriel A DeAvila, Ariosto S Silva, Mark B Meads, Yvonne Parker, Daniel J Lindner, Yogen Saunthararajah, Kenneth H Shain, Jaroslaw P Maciejewski, Frederic J Reu, James G Phillips, Babal K Jha.

  Multiple myeloma is a genetically complex hematologic neoplasia in which malignant plasma cells constantly operate at the maximum limit of their unfolded protein response (UPR) due to a high secretory burden of immunoglobulins and cytokines. The endoplasmic reticulum (ER) resident protein disulfide isomerase, PDIA1 is indispensable for maintaining structural integrity of cysteine-rich antibodies and cytokines that require accurate intramolecular disulfide bond arrangement. PDIA1 expression analysis from RNA-seq of multiple myeloma patients demonstrated an inverse relationship with survival in relapsed or refractory disease, supporting its critical role in myeloma persistence. Using a structure-guided medicinal chemistry approach, we developed a potent, orally bioavailable small molecule PDIA1 inhibitor CCF642-34. The inhibition of PDIA1 overwhelms the UPR in myeloma cells, resulting in their apoptotic cell death at doses that do not affect the normal CD34+ hematopoietic stem and progenitor cells. Bortezomib resistance leads to increased PDIA1 expression and thus CCF642-34 sensitivity, suggesting that proteasome inhibitor resistance leads to PDIA1 dependence for proteostasis and survival. CCF642-34 induces acute unresolvable UPR in myeloma cells, and oral treatment increased survival of mice in the syngeneic 5TGM1 model of myeloma. Results support development of CCF642-34 to selectively target the plasma cell program and overcome the treatment-refractory state in myeloma.

Keywords:  ER stress; ERMM; IRMM; UPR; protein disulfide isomerase PDIA1

DOI:  https://doi.org/10.3390/cancers13112649
J Vis Exp. 2021 May 14.

In Vitro Analysis of E3 Ubiquitin Ligase Function.

Leonie Müller, Carl Elias Kutzner, Vishnu Balaji, Thorsten Hoppe.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

DOI: https://doi.org/10.3791/62393
Biochim Biophys Acta Proteins Proteom. 2021 May 27. pii: S1570-9639(21)00086-8. [Epub ahead of print]1869(9): 140680

HSPB1 influences mitochondrial respiration in ER-stressed beta cells.

Simon Ngao Mule, Vinícius De Morais Gomes, Rosangela A M Wailemann, Janaina Macedo-da-Silva, Livia Rosa-Fernandes, Martin R Larsen, Letícia Labriola, Giuseppe Palmisano.

  Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.

Keywords:  Diabetes; ER stress; HSPB1; Mass spectrometry; Mitochondrial bioenergetics; Pancreatic beta cells; Proteomics; UPR

DOI:  https://doi.org/10.1016/j.bbapap.2021.140680
J Cell Sci. 2021 Jun 01. pii: jcs258338. [Epub ahead of print]134(11):

Genome-wide imaging screen uncovers molecular determinants of arsenite-induced protein aggregation and toxicity.

Stefanie Andersson, Antonia Romero, Joana Isabel Rodrigues, Sansan Hua, Xinxin Hao, Therese Jacobson, Vivien Karl, Nathalie Becker, Arghavan Ashouri, Sebastien Rauch, Thomas Nyström, Beidong Liu, Markus J Tamás.

  The toxic metalloid arsenic causes widespread misfolding and aggregation of cellular proteins. How these protein aggregates are formed in vivo, the mechanisms by which they affect cells and how cells prevent their accumulation is not fully understood. To find components involved in these processes, we performed a genome-wide imaging screen and identified Saccharomyces cerevisiae deletion mutants with either enhanced or reduced protein aggregation levels during arsenite exposure. We show that many of the identified factors are crucial to safeguard protein homeostasis (proteostasis) and to protect cells against arsenite toxicity. The hits were enriched for various functions including protein biosynthesis and transcription, and dedicated follow-up experiments highlight the importance of accurate transcriptional and translational control for mitigating protein aggregation and toxicity during arsenite stress. Some of the hits are associated with pathological conditions, suggesting that arsenite-induced protein aggregation may affect disease processes. The broad network of cellular systems that impinge on proteostasis during arsenic stress identified in this current study provides a valuable resource and a framework for further elucidation of the mechanistic details of metalloid toxicity and pathogenesis. This article has an associated First Person interview with the first authors of the paper.

Keywords:  Arsenic; Protein aggregation; Protein misfolding; Protein quality control; Proteostasis; Transcription; Translation; Yeast

DOI:  https://doi.org/10.1242/jcs.258338
PLoS Biol. 2021 Jun 02. 19(6): e3001281

The nonreceptor tyrosine kinase SRMS inhibits autophagy and promotes tumor growth by phosphorylating the scaffolding protein FKBP51.

Jung Mi Park, Seung Wook Yang, Wei Zhuang, Asim K Bera, Yan Liu, Deepak Gurbani, Sergei J von Hoyningen-Huene, Sadie Miki Sakurada, Haiyun Gan, Shondra M Pruett-Miller, Kenneth D Westover, Malia B Potts.

Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates.

DOI: https://doi.org/10.1371/journal.pbio.3001281
Cancers (Basel). 2021 May 20. pii: 2490. [Epub ahead of print]13(10):

The Carcinogen Cadmium Activates Lysine 63 (K63)-Linked Ubiquitin-Dependent Signaling and Inhibits Selective Autophagy.

Abderrahman Chargui, Amine Belaid, Papa Diogop Ndiaye, Véronique Imbert, Michel Samson, Jean-Marie Guigonis, Michel Tauc, Jean-François Peyron, Philippe Poujeol, Patrick Brest, Paul Hofman, Baharia Mograbi.

  Signaling, proliferation, and inflammation are dependent on K63-linked ubiquitination-conjugation of a chain of ubiquitin molecules linked via lysine 63. However, very little information is currently available about how K63-linked ubiquitination is subverted in cancer. The present study provides, for the first time, evidence that cadmium (Cd), a widespread environmental carcinogen, is a potent activator of K63-linked ubiquitination, independently of oxidative damage, activation of ubiquitin ligase, or proteasome impairment. We show that Cd induces the formation of protein aggregates that sequester and inactivate cylindromatosis (CYLD) and selective autophagy, two tumor suppressors that deubiquitinate and degrade K63-ubiquitinated proteins, respectively. The aggregates are constituted of substrates of selective autophagy-SQSTM1, K63-ubiquitinated proteins, and mitochondria. These protein aggregates also cluster double-membrane remnants, which suggests an impairment in autophagosome maturation. However, failure to eliminate these selective cargos is not due to alterations in the general autophagy process, as degradation of long-lived proteins occurs normally. We propose that the simultaneous disruption of CYLD and selective autophagy by Cd feeds a vicious cycle that further amplifies K63-linked ubiquitination and downstream activation of the NF-κB pathway, processes that support cancer progression. These novel findings link together impairment of selective autophagy, K63-linked ubiquitination, and carcinogenesis.

Keywords:  CYLD deubiquitinase; K63-linked ubiquitination; NF-κB; aggrephagy; bafilomycin A1; cadmium; carcinogen; selective autophagy; starvation-induced autophagy; tumor suppression

DOI:  https://doi.org/10.3390/cancers13102490
FEBS J. 2021 Jun 05.

The interplay between SUMOylation and phosphorylation of PKCδ facilitates oxidative stress-induced apoptosis.

Siman Gao, Xiangteng Zhao, Lin Hou, Ruining Ma, Jie Zhou, Michael X Zhu, Si-Jian Pan, Yong Li.

  Although the increase in the number of identified posttranslational modifications (PTMs) has substantially improved our knowledge about substrate site specificity of single PTMs, the fact that different types of PTMs can crosstalk and act in concert to exert important regulatory mechanisms for protein function has not gained much attention. Here, we show that PKCδ is SUMOylated at lysine 473 in its C-terminal catalytic domain, and the SUMOylation increases PKCδ stability by repressing its ubiquitination. In addition, we uncover a functional interplay between the phosphorylation and SUMOylation of PKCδ, which can strengthen each other through recruiting SUMO E2/E3 ligases and the PKCδ kinase, respectively, to the PKCδ complexes. We identified PIAS2β as the SUMO E3 ligase of PKCδ. More importantly, by enhancing PKCδ protein stability and its phosphorylation through an interdependent interplay of the PTMs, the SUMOylation of PKCδ promotes apoptotic cell death induced by H2 O2 . We conclude that SUMOylation represents an important regulatory mechanism of PKCδ PTMs for the kinase's function in oxidative cell damage.

Keywords:  Apoptosis; Oxidative damage; PKCδ SUMOylation; PKCδ degradation; PKCδ phosphorylation

DOI:  https://doi.org/10.1111/febs.16050
Cell Mol Life Sci. 2021 Jun 05.

ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs.

Yevgen Zolotarov, Chao Ma, Irene González-Recio, Serge Hardy, Gijs A C Franken, Noriko Uetani, Femke Latta, Elie Kostantin, Jonathan Boulais, Marie-Pier Thibault, Jean-François Côté, Irene Díaz Moreno, Antonio Díaz Quintana, Joost G J Hoenderop, Luis Alfonso Martínez-Cruz, Michel L Tremblay, Jeroen H F de Baaij.

  Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-β-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.

Keywords:  CNNM2; CNNM3; Glycosylation; Magnesium transport; Protein-protein interaction

DOI:  https://doi.org/10.1007/s00018-021-03832-8
Cancers (Basel). 2021 May 08. pii: 2260. [Epub ahead of print]13(9):

Alterations of the Platelet Proteome in Lung Cancer: Accelerated F13A1 and ER Processing as New Actors in Hypercoagulability.

Huriye Ercan, Lisa-Marie Mauracher, Ella Grilz, Lena Hell, Roland Hellinger, Johannes A Schmid, Florian Moik, Cihan Ay, Ingrid Pabinger, Maria Zellner.

  In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins calreticulin (CALR), endoplasmic reticulum chaperone BiP (HSPA5) and protein disulfide-isomerase (P4HB) were significantly elevated. Accelerated conversion of the fibrin stabilising factor XIII was detected in platelets of patients with lung cancer by elevated levels of a coagulation factor XIII (F13A1) 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma levels of the plasmin-α-2-antiplasmin complex and D-dimer suggests its enhanced degradation by the fibrinolytic system. Protein association network analysis showed that lung cancer-related proteins were involved in platelet degranulation and upregulated ER protein processing. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Keywords:  cancer; coagulation factor XIII; endoplasmic reticulum chaperones; lung cancer; platelets; protein disulfide-isomerase; proteomics; therapeutic target; thrombosis; unfolded protein response

DOI:  https://doi.org/10.3390/cancers13092260
In Vitro Cell Dev Biol Anim. 2021 Jun 03.

UFL1 regulates milk protein and fat synthesis-related gene expression of bovine mammary epithelial cells probably via the mTOR signaling pathway.

Chengmin Li, Lian Li, Ilyas Ali, Meiqian Kuang, Xinling Wang, Genlin Wang.

  UFL1 is an ufmylation (a novel post-translational modification) E3 ligase, mainly located in the endoplasmic reticulum (ER), that has emerged as a significant regulator of several physiological and pathological processes. Yet its physiological function in milk synthesis in bovine mammary epithelial cells (BMECs) remains unknown. In this study, we investigated the effects of UFL1 in milk protein and fat synthesis-related gene expression, with a particular emphasis on the role of UFL1 in LPS-treated BMECs. Results showed that UFL1 depletion significantly reduced the expression of milk protein and fat synthesis-related gene and mTOR phosphorylation in both normal and LPS-treated BMECs. Overexpression of UFL1 enhanced the activation of the mTOR and milk protein and fat synthesis-related gene expression. Collectively, these above results strongly demonstrate that UFL1 could regulate milk protein and fat synthesis-related gene expression of BMECs probably via the mTOR signaling pathway.

Keywords:  Bovine mammary epithelial cells; Cell proliferation; Milk synthesis; UFL1; mTOR

DOI:  https://doi.org/10.1007/s11626-021-00587-1
Antioxid Redox Signal. 2021 Jun 02.

ERO1-PDI redox signaling in health and disease.

Vishwanath Jha, Tripti Kumari, Vijayprakash Manickam, Zahra Assar, Kirk L Olson, Jeong-Ki Min, Jaehyung Cho.

SIGNIFICANCE: Protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductase 1 (ERO1) are crucial for oxidative protein folding in the ER. These enzymes are frequently overexpressed and secreted and contribute to the pathology of neurodegenerative, cardiovascular, and metabolic diseases. Recent Advances: Tissue-specific knockout mouse models and pharmacologic inhibitors have been developed to advance our understanding of the cell-specific functions of PDI and ERO1. In addition to their roles in protecting cells from the unfolded protein response and oxidative stress, recent studies have revealed that PDI and ERO1 also regulate extracellular functions and activities.
CRITICAL ISSUES: Despite the well-known contributions of PDI and ERO1 to specific disease pathology, the detailed molecular and cellular mechanisms underlying these activities remain to be elucidated. Furthermore, while PDI and ERO1 inhibitors have been identified, the results from previous studies require careful evaluation, as many of these agents are not selective and may have significant cytotoxicity.
FUTURE DIRECTIONS: The function of PDI and ERO1 in the ER have been studied extensively. Additional studies will be required to define their functions outside the ER.

DOI: https://doi.org/10.1089/ars.2021.0018
Int J Mol Sci. 2021 May 25. pii: 5584. [Epub ahead of print]22(11):

Ubiquitin-Specific Protease 3 Deubiquitinates and Stabilizes Oct4 Protein in Human Embryonic Stem Cells.

Byung-Ho Rhie, Ainsley Mike Antao, Janardhan Keshav Karapurkar, Min-Seong Kim, Won-Jun Jo, Suresh Ramakrishna, Kye-Seong Kim.

  Oct4 is an important mammalian POU family transcription factor expressed by early human embryonic stem cells (hESCs). The precise level of Oct4 governs the pluripotency and fate determination of hESCs. Several post-translational modifications (PTMs) of Oct4 including phosphorylation, ubiquitination, and SUMOylation have been reported to regulate its critical functions in hESCs. Ubiquitination and deubiquitination of Oct4 should be well balanced to maintain the pluripotency of hESCs. The protein turnover of Oct4 is regulated by several E3 ligases through ubiquitin-mediated degradation. However, reversal of ubiquitination by deubiquitinating enzymes (DUBs) has not been reported for Oct4. In this study, we generated a ubiquitin-specific protease 3 (USP3) gene knockout using the CRISPR/Cas9 system and demonstrated that USP3 acts as a protein stabilizer of Oct4 by deubiquitinating Oct4. USP3 interacts with endogenous Oct4 and co-localizes in the nucleus of hESCs. The depletion of USP3 leads to a decrease in Oct4 protein level and loss of pluripotent morphology in hESCs. Thus, our results show that USP3 plays an important role in controlling optimum protein level of Oct4 to retain pluripotency of hESCs.

Keywords:  26S proteasome; CRISPR/Cas9; embryonic carcinoma cells; gene knockout; post-translational modifications

DOI:  https://doi.org/10.3390/ijms22115584
Cell Chem Biol. 2021 May 26. pii: S2451-9456(21)00222-1. [Epub ahead of print]

Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex.

Jong Hee Song, Nicole D Wagner, Jing Yan, Jing Li, Richard Y-C Huang, Aaron J Balog, John A Newitt, Guodong Chen, Michael L Gross.

  Proteolysis-targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein of interest (POI) induces its ubiquitination-based proteolysis by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of an E3 ubiquitin ligase) promotes POI dissociation. Collision-induced dissociation (CID) causes elimination of the nonperipheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. In addition, we used ion mobility spectrometry (IMS) and collisional activation to identify the source of this unexpected dissociation. Together, the evidence shows that this integrated approach can be used to screen for ternary complex formation and PROTAC-protein contacts and may report on PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy.

Keywords:  CID (collision-induced dissociation); CIU (collision-induced unfolding); E3 ligase; ECD (electron capture dissociation); IMS (ion mobility spectrometry); MZ1; PROTACs (proteolysis-targeting chimeras); charge-reducing agent; native mass spectrometry; ternary complex

DOI:  https://doi.org/10.1016/j.chembiol.2021.05.005
Nature. 2021 Jun 02.

A proximity-dependent biotinylation map of a human cell.

Christopher D Go, James D R Knight, Archita Rajasekharan, Bhavisha Rathod, Geoffrey G Hesketh, Kento T Abe, Ji-Young Youn, Payman Samavarchi-Tehrani, Hui Zhang, Lucie Y Zhu, Evelyn Popiel, Jean-Philippe Lambert, Étienne Coyaud, Sally W T Cheung, Dushyandi Rajendran, Cassandra J Wong, Hana Antonicka, Laurence Pelletier, Alexander F Palazzo, Eric A Shoubridge, Brian Raught, Anne-Claude Gingras.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

DOI: https://doi.org/10.1038/s41586-021-03592-2
FEBS Lett. 2021 May 31.

MED12 interacts with the heat shock transcription factor HSF1 and recruits CDK8 to promote the heat shock response in mammalian cells.

Pratibha Srivastava, Ryosuke Takii, Mariko Okada, Mitsuaki Fujimoto, Akira Nakai.

  Activated and promoter-bound heat shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1-mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12, and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1-S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress.

Keywords:  CDK8; HSF1; MED12; heat shock; phosphorylation; transcription

DOI:  https://doi.org/10.1002/1873-3468.14139
Int J Mol Sci. 2021 May 23. pii: 5488. [Epub ahead of print]22(11):

Ubiquitination and Deubiquitination in Oral Disease.

Sachio Tsuchida, Tomohiro Nakayama.

  Oral health is an integral part of the general health and well-being of individuals. The presence of oral disease is potentially indicative of a number of systemic diseases and may contribute to their early diagnosis and treatment. The ubiquitin (Ub) system has been shown to play a role in cellular immune response, cellular development, and programmed cell death. Ubiquitination is a post-translational modification that occurs in eukaryotes. Its mechanism involves a number of factors, including Ub-activating enzymes, Ub-conjugating enzymes, and Ub protein ligases. Deubiquitinating enzymes, which are proteases that reversely modify proteins by removing Ub or Ub-like molecules or remodeling Ub chains on target proteins, have recently been regarded as crucial regulators of ubiquitination-mediated degradation and are known to significantly affect cellular pathways, a number of biological processes, DNA damage response, and DNA repair pathways. Research has increasingly shown evidence of the relationship between ubiquitination, deubiquitination, and oral disease. This review investigates recent progress in discoveries in diseased oral sites and discusses the roles of ubiquitination and deubiquitination in oral disease.

Keywords:  Ubiquitination; deubiquitinating enzymes; deubiquitination; oral disease; ubiquitin-conjugating enzyme

DOI:  https://doi.org/10.3390/ijms22115488
J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00518-4. [Epub ahead of print] 100729

Calcium-dependent and -independent lipid transfer mediated by tricalbins in yeast.

Tiantian Qian, Chenlu Li, Ruyue He, Chun Wan, Yinghui Liu, Haijia Yu.

  Membrane contact sites (MCSs) formed between the endoplasmic reticulum (ER) and the plasma membrane (PM) provide a platform for non-vesicular lipid exchange. The ER-anchored tricalbins (Tcb1, Tcb2, and Tcb3) are critical tethering factors at ER-PM MCSs in yeast. Tricalbins possess a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and multiple Ca2+-binding C2 domains. Although tricalbins have been suggested to be involved in lipid exchange at the ER-PM MCSs, it remains unclear whether they directly mediate lipid transport. Here, using in vitro lipid transfer assays, we discovered that tricalbins are capable of transferring phospholipids between membranes. Unexpectedly, while its lipid transfer activity was markedly elevated by Ca2+, Tcb3 constitutively transferred lipids even in the absence of Ca2+. The stimulatory activity of Ca2+ on Tcb3 required intact Ca2+-binding sites on both the C2C and C2D domains of Tcb3, while Ca2+-independent lipid transport was mediated by the SMP domain that transferred lipids via direct interactions with phosphatidylserine and other negatively charged lipid molecules. These findings establish tricalbins as lipid transfer proteins, and reveal Ca2+-dependent and -independent lipid transfer activities mediated by these tricalbins, providing new insights into their mechanism in maintaining PM integrity at ER-PM MCSs.

Keywords:  SMP; endoplasmic reticulum; lipid transfer; membrane contact sites; plasma membrane; tricalbin

DOI:  https://doi.org/10.1016/j.jbc.2021.100729
Biochem J. 2021 Jun 01. pii: BCJ20201000. [Epub ahead of print]

The parkinsonism-associated protein FBXO7 cooperates with the BAG6 complex in proteasome function and controls the subcellular localization of the complex.

Quan Wang, Vanessa Crnković, Christian Preisinger, Judith Stegmüller.

  The regulation of proteasome activity is essential to cellular homeostasis and defects have been implicated in various disorders including Parkinson disease. The F-box protein FBXO7 has been implicated in early-onset parkinsonism and has previously been shown to have a regulatory role in proteasome activity and assembly. Here, we report the association of the E3 ubiquitin ligase FBXO7-SCF (SKP1, cullin-1, F-box protein) with the BAG6 complex, consisting of the subunits BAG6, GET4 and UBL4A. We identify the subunit GET4 as a direct interactor of FBXO7 and we show that the subunits GET4 and UBL4A are required for proper proteasome activity. Our findings demonstrate reduced binding of FBXO7 variants to GET4 and that FBXO7 variants bring about reduced proteasome activity. In addition, we find that GET4 is a non-proteolytic substrate of FBXO7, that binding of GET4 to BAG6 is enhanced in the presence of active FBXO7-SCF and that the cytoplasmic localization of the BAG6 complex is dependent on the E3 ubiquitin ligase activity. Taken together, our study shows that the parkinsonism-associated FBXO7 cooperates with the BAG6 complex in proteasome function and determines the subcellular localization of this complex.

Keywords:  E3 ubiquitin ligase; Parkinsons disease; proteasome; ubiquitination

DOI:  https://doi.org/10.1042/BCJ20201000
J Cell Sci. 2021 Jun 01. pii: jcs.249771. [Epub ahead of print]

SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A.

Bin Yu, Qiaoyu Lin, Chao Huang, Boyan Zhang, Ying Wang, Qing Jiang, Chuanmao Zhang, Jing Yi.

  Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator, Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupted the deSUMOylation of AurA, leading to an increased kinase activity and abnormalities in spindle assembly and chromosomes segregation which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render a spatiotemporal control on its kinase activity in mitosis.

Keywords:  Aurora A; Mitosis; SENP3; SENP5; SUMOylation; Spindle assembly

DOI:  https://doi.org/10.1242/jcs.249771
Mol Cell. 2021 Jun 03. pii: S1097-2765(21)00360-9. [Epub ahead of print]81(11): 2270-2272

The GID ubiquitin ligase complex just reached the next level of complexity.

Lisa Fechtner, Thorsten Pfirrmann.

This issue of Molecular Cell features two publications that make striking discoveries concerning the GID ubiquitin ligase complex. Kong et al. (2021) describe a substrate recognition mechanism, expanding the set of GID complex substrates, whereas Sherpa et al. (2021) unravel the molecular mechanism by which the GID complex targets the quaternary structure of a substrate.

DOI: https://doi.org/10.1016/j.molcel.2021.05.003
BMC Biol. 2021 Jun 04. 19(1): 117

Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase.

Cheng-Wen He, Xue-Fei Cui, Shao-Jie Ma, Qin Xu, Yan-Peng Ran, Wei-Zhi Chen, Jun-Xi Mu, Hui Li, Jing Zhu, Qingqiu Gong, Zhiping Xie.

   BACKGROUND: The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways.
RESULTS: Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation.
CONCLUSIONS: Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

Keywords:  Atg8; ESCRT; Hfl1; Microautophagy; Vacuole; Yeast

DOI:  https://doi.org/10.1186/s12915-021-01048-7
Proteomics. 2021 Jun 04. e2000234

Modification of small ubiquitin-related modifier 2 (SUMO2) by phosphoubiquitin in HEK293T cells.

Julius T Dongdem, Simon P Dawson, Robert Layfield.

  Additional complexity in the post-translational modification of proteins by ubiquitin is achieved by ubiquitin phosphorylation, for example within PINK1-parkin mediated mitophagy. We performed a preliminary proteomic analysis to identify proteins differentially modified by ubiquitin in HEK293T, compared to phosphomimetic ubiquitin (Ser65Asp), and identified SUMO2 as a candidate. By transfecting SUMO2 and its C-terminal-GG deletion mutant, along with phosphomimetic ubiquitin, we confirm that ubiquitin modifies SUMO2, rather than vice versa. Further investigations revealed that transfected SUMO2 can also be conjugated by endogenous phospho-Ser65-(poly)ubiquitin in HEK293T cells, pointing to a previously unappreciated level of complexity in SUMO2 modification, and that unanchored (substrate-free) polyubiquitin chains may also be subject to phosphorylation. This article is protected by copyright. All rights reserved.

Keywords:  HEK293T; Phosphorylation; Post-translational modification; SUMO2; Ser65; Ubiquitin; Unanchored polyubiquitin

DOI:  https://doi.org/10.1002/pmic.202000234
Cell Chem Biol. 2021 May 22. pii: S2451-9456(21)00223-3. [Epub ahead of print]

Selective autophagy as the basis of autophagy-based degraders.

Daiki Takahashi, Hirokazu Arimoto.

  Degrader technologies, which enable the chemical knockdown of disease-causing proteins, are promising for drug discovery. After two decades of research, degraders using the ubiquitin-proteasome system (UPS) are currently in clinical trials. However, the UPS substrates are mainly limited to soluble proteins. Autophagy-targeting chimeras and autophagosome-tethering compounds are degraders that use autophagy, which has functions complementary to the UPS. They can degrade organelles and aggregate-prone proteins, making them promising treatments against age-related conditions such as mitochondrial dysfunction and neurodegenerative diseases. The molecular mechanism of selective autophagy is an ongoing research topic, which explains why autophagy-based degraders were not available until recently. In this review, we introduce four classifications of selective autophagy mechanisms to facilitate the understanding of the degrader design.

Keywords:  ATTEC; AUTAC; LLPS; S-guanylation; aggregates; autophagy; degrader; mitochondria; p62; ubiquitin

DOI:  https://doi.org/10.1016/j.chembiol.2021.05.006
Trends Biochem Sci. 2021 May 27. pii: S0968-0004(21)00103-1. [Epub ahead of print]

Origins and evolving functionalities of tRNA-derived small RNAs.

Qi Chen, Xudong Zhang, Junchao Shi, Menghong Yan, Tong Zhou.

  Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are among the most ancient small RNAs in all domains of life and are generated by the cleavage of tRNAs. Emerging studies have begun to reveal the versatile roles of tsRNAs in fundamental biological processes, including gene silencing, ribosome biogenesis, retrotransposition, and epigenetic inheritance, which are rooted in tsRNA sequence conservation, RNA modifications, and protein-binding abilities. We summarize the mechanisms of tsRNA biogenesis and the impact of RNA modifications, and propose how thinking of tsRNA functionality from an evolutionary perspective urges the expansion of tsRNA research into a wider spectrum, including cross-tissue/cross-species regulation and harnessing of the 'tsRNA code' for precision medicine.

Keywords:  Argonaute proteins; mimicry; ribonucleoprotein (RNP); ribozyme; tRNA fragmentation; translation

DOI:  https://doi.org/10.1016/j.tibs.2021.05.001
Cell Death Discov. 2021 May 31. 7(1): 128

P300/HDAC1 regulates the acetylation/deacetylation and autophagic activities of LC3/Atg8-PE ubiquitin-like system.

Wenmei Wu, Kang Li, Sanyou Guo, Jing Xu, Qiuqin Ma, Shuyan Li, Xianying Xu, Zhijun Huang, Yangjin Zhong, Gianluca Tettamanti, Yang Cao, Sheng Li, Ling Tian.

Protein acetylation plays potential roles in regulating autophagy occurrence. However, it varies greatly between yeast and mammals, and has not been thoroughly investigated in other organisms. Here, we reported that the components of BmAtg8-PE ubiquitin-like system (BmAtg3, BmAtg4, BmAtg7, and BmAtg8) in Bombyx mori were localized in the nucleus under nutrient-rich conditions, whereas they were exported to the cytoplasm upon autophagy induction. RNAi of BmP300 and inhibition of BmP300 activity resulted in nucleo-cytoplasmic translocation of BmAtg3 and BmAtg8, as well as premature induction of autophagy in the absence of stimulus. Conversely, RNAi of BmHDAC1 and inhibition of class I/II HADCs activities led to the nuclear accumulation of BmAtg3 and BmAtg8. In addition, acetylation sites in Atg proteins of BmAtg8-PE ubiquitin-like system were identified by mass spectrometry, and acetylation-site mutations caused nucleo-cytoplasmic translocation of BmAtg3, BmAtg4, and BmAtg8 along with autophagy promotion. Similarly, the subcellular localization of human ATG4b is determined by acetylation modification. In general, BmP300-mediated acetylation sequesters the components of BmAtg8-PE ubiquitin-like system in the nucleus, thus leading to the autophagy inhibition. Oppositely, BmHDAC1-mediated deacetylation leads to the nucleo-cytoplasmic translocation of the components of BmAtg8-PE ubiquitin-like system and promotes autophagy. This process is evolutionarily conserved between insects and mammals.

DOI: https://doi.org/10.1038/s41420-021-00513-0
Elife. 2021 Jun 04. pii: e64140. [Epub ahead of print]10

Short and long sleeping mutants reveal links between sleep and macroautophagy.

Joseph L Bedont, Hirofumi Toda, Mi Shi, Christine H Park, Christine Quake, Carly Stein, Anna Kolesnik, Amita Sehgal.

  Sleep is a conserved and essential behavior, but its mechanistic and functional underpinnings remain poorly defined. Through unbiased genetic screening in Drosophila, we discovered a novel short-sleep mutant we named argus. Positional cloning and subsequent complementation, CRISPR/Cas9 knock-out, and RNAi studies identified Argus as a transmembrane protein that acts in adult peptidergic neurons to regulate sleep. argus mutants accumulate undigested Atg8a(+) autophagosomes, and genetic manipulations impeding autophagosome formation suppress argus sleep phenotypes, indicating that autophagosome accumulation drives argus short-sleep. Conversely, a blue cheese neurodegenerative mutant that impairs autophagosome formation was identified independently as a gain-of-sleep mutant, and targeted RNAi screens identified additional genes involved in autophagosome formation whose knockdown increases sleep. Finally, autophagosomes normally accumulate during the daytime and nighttime sleep deprivation extends this accumulation into the following morning, while daytime gaboxadol feeding promotes sleep and reduces autophagosome accumulation at nightfall. In sum, our results paradoxically demonstrate that wakefulness increases and sleep decreases autophagosome levels under unperturbed conditions, yet strong and sustained upregulation of autophagosomes decreases sleep, whereas strong and sustained downregulation of autophagosomes increases sleep. The complex relationship between sleep and autophagy suggested by our findings may have implications for pathological states including chronic sleep disorders and neurodegeneration, as well as for integration of sleep need with other homeostats, such as under conditions of starvation.

Keywords:  D. melanogaster; Drosophila; argus; autophagy; blue cheese; cell biology; genetics; neuroscience; sleep

DOI:  https://doi.org/10.7554/eLife.64140
Trends Immunol. 2021 May 29. pii: S1471-4906(21)00098-3. [Epub ahead of print]

OTULIN in NF-κB signaling, cell death, and disease.

Lien Verboom, Esther Hoste, Geert van Loo.

Tight control of inflammatory signaling pathways is an absolute requirement to avoid chronic inflammation and disease. One of the proteins responsible for such control is OTU deubiquitinase with linear linkage specificity (OTULIN), the only mammalian deubiquitinating enzyme (DUB) exclusively hydrolyzing linear ubiquitin chains from proteins modified by the linear ubiquitin chain assembly complex (LUBAC) described thus far. Recent findings show that loss-of-function mutations in OTULIN underlie a severe early-onset human autoinflammatory disease and severe pathology in experimental mouse models. Here, we review the molecular and cellular mechanisms by which OTULIN controls inflammation and discuss the involvement of OTULIN in inflammatory disease development. We also highlight several newly identified roles for OTULIN, including a ubiquitin-independent function.

DOI: https://doi.org/10.1016/j.it.2021.05.003
Nat Commun. 2021 05 31. 12(1): 3263

Cryptic genetic variation in a heat shock protein modifies the outcome of a mutation affecting epidermal stem cell development in C. elegans.

Sneha L Koneru, Mark Hintze, Dimitris Katsanos, Michalis Barkoulas.

A fundamental question in medical genetics is how the genetic background modifies the phenotypic outcome of mutations. We address this question by focusing on the seam cells, which display stem cell properties in the epidermis of Caenorhabditis elegans. We demonstrate that a putative null mutation in the GATA transcription factor egl-18, which is involved in seam cell fate maintenance, is more tolerated in the CB4856 isolate from Hawaii than the lab reference strain N2 from Bristol. We identify multiple quantitative trait loci (QTLs) underlying the difference in phenotype expressivity between the two isolates. These QTLs reveal cryptic genetic variation that reinforces seam cell fate through potentiating Wnt signalling. Within one QTL region, a single amino acid deletion in the heat shock protein HSP-110 in CB4856 is sufficient to modify Wnt signalling and seam cell development, highlighting that natural variation in conserved heat shock proteins can shape phenotype expressivity.

DOI: https://doi.org/10.1038/s41467-021-23567-1
Nat Med. 2021 May 31.

Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.

Payam Mohassel, Sandra Donkervoort, Museer A Lone, Matthew Nalls, Kenneth Gable, Sita D Gupta, A Reghan Foley, Ying Hu, Jonas Alex Morales Saute, Ana Lucila Moreira, Fernando Kok, Alessandro Introna, Giancarlo Logroscino, Christopher Grunseich, Alec R Nickolls, Naemeh Pourshafie, Sarah B Neuhaus, Dimah Saade, Andrea Gangfuß, Heike Kölbel, Zoe Piccus, Claire E Le Pichon, Chiara Fiorillo, Cindy V Ly, Ana Töpf, Lauren Brady, Sabine Specht, Aliza Zidell, Helio Pedro, Eric Mittelmann, Florian P Thomas, Katherine R Chao, Chamindra G Konersman, Megan T Cho, Tracy Brandt, Volker Straub, Anne M Connolly, Ulrike Schara, Andreas Roos, Mark Tarnopolsky, Ahmet Höke, Robert H Brown, Chia-Hsueh Lee, Thorsten Hornemann, Teresa M Dunn, Carsten G Bönnemann.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.

DOI: https://doi.org/10.1038/s41591-021-01346-1
Cells. 2021 May 18. pii: 1235. [Epub ahead of print]10(5):

To Ubiquitinate or Not to Ubiquitinate: TRIM17 in Cell Life and Death.

Meenakshi Basu-Shrivastava, Alina Kozoriz, Solange Desagher, Iréna Lassot.

  TRIM17 is a member of the TRIM family, a large class of RING-containing E3 ubiquitin-ligases. It is expressed at low levels in adult tissues, except in testis and in some brain regions. However, it can be highly induced in stress conditions which makes it a putative stress sensor required for the triggering of key cellular responses. As most TRIM members, TRIM17 can act as an E3 ubiquitin-ligase and promote the degradation by the proteasome of substrates such as the antiapoptotic protein MCL1. Intriguingly, TRIM17 can also prevent the ubiquitination of other proteins and stabilize them, by binding to other TRIM proteins and inhibiting their E3 ubiquitin-ligase activity. This duality of action confers several pivotal roles to TRIM17 in crucial cellular processes such as apoptosis, autophagy or cell division, but also in pathological conditions as diverse as Parkinson's disease or cancer. Here, in addition to recent data that endorse this duality, we review what is currently known from public databases and the literature about TRIM17 gene regulation and expression, TRIM17 protein structure and interactions, as well as its involvement in cell physiology and human disorders.

Keywords:  Parkinson’s disease; TRIM17; apoptosis; autism; autophagy; cancer; mitosis; proteolysis; ubiquitination

DOI:  https://doi.org/10.3390/cells10051235
Hum Mol Genet. 2021 May 31. pii: ddab145. [Epub ahead of print]

Variants in USP48 encoding ubiquitin hydrolase are associated with autosomal dominant non-syndromic hereditary hearing loss.

Sissy Bassani, Edward Beelen, Mireille Rossel, Norine Voisin, Anna Morgan, Yoan Arribat, Nicolas Chatron, Jacqueline Chrast, Massimiliano Cocca, Benjamin Delprat, Flavio Faletra, Giuliana Giannuzzi, Nicolas Guex, Roxane Machavoine, Sylvain Pradervand, Jeroen J Smits, Jiddeke M van de Kamp, Alban Ziegler, Francesca Amati, Sandrine Marlin, Hannie Kremer, Heiko Locher, Tangui Maurice, Paolo Gasparini, Giorgia Girotto, Alexandre Reymond.

Non-Syndromic Hereditary Hearing Loss (NSHHL) is a genetically heterogeneous sensory disorder with about 120 genes already associated. Through exome sequencing and data aggregation, we identified a family with six affected individuals and one unrelated NSHHL patient with predicted-to-be deleterious missense variants in USP48. We also uncovered an eighth patient presenting unilateral cochlear nerve aplasia and a de novo splice variant in the same gene. USP48 encodes a ubiquitin carboxyl-terminal hydrolase under evolutionary constraint. Pathogenicity of the variants is supported by in vitro assays that showed that the mutated proteins are unable to hydrolyze tetra-ubiquitin. Correspondingly, three-dimensional representation of the protein containing the familial missense variant affects a loop that controls binding to ubiquitin. Consistent with a contribution of USP48 to auditory function, immunohistology showed that the encoded protein is expressed in the developing human inner ear, specifically in the spiral ganglion neurons, outer sulcus, interdental cells of the spiral limbus, stria vascularis, Reissner's membrane, and in the transient Kolliker's organ that is essential for auditory development. Engineered zebrafish knocked-down for usp48, the USP48 ortholog, presented with a delayed development of primary motor neurons, less developed statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Corroboratingly, acoustic startle response assays revealed a significant decrease of auditory response of zebrafish lacking usp48 at 600 Hz and 800 Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining exome sequencing, animal modeling, immunohistology and molecular assays.

DOI: https://doi.org/10.1093/hmg/ddab145
Nat Commun. 2021 06 02. 12(1): 3291

UBE4B, a microRNA-9 target gene, promotes autophagy-mediated Tau degradation.

Manivannan Subramanian, Seung Jae Hyeon, Tanuza Das, Yoon Seok Suh, Yun Kyung Kim, Jeong-Soo Lee, Eun Joo Song, Hoon Ryu, Kweon Yu.

The formation of hyperphosphorylated intracellular Tau tangles in the brain is a hallmark of Alzheimer's disease (AD). Tau hyperphosphorylation destabilizes microtubules, promoting neurodegeneration in AD patients. To identify suppressors of tau-mediated AD, we perform a screen using a microRNA (miR) library in Drosophila and identify the miR-9 family as suppressors of human tau overexpression phenotypes. CG11070, a miR-9a target gene, and its mammalian orthologue UBE4B, an E3/E4 ubiquitin ligase, alleviate eye neurodegeneration, synaptic bouton defects, and crawling phenotypes in Drosophila human tau overexpression models. Total and phosphorylated Tau levels also decrease upon CG11070 or UBE4B overexpression. In mammalian neuroblastoma cells, overexpression of UBE4B and STUB1, which encodes the E3 ligase CHIP, increases the ubiquitination and degradation of Tau. In the Tau-BiFC mouse model, UBE4B and STUB1 overexpression also increase oligomeric Tau degradation. Inhibitor assays of the autophagy and proteasome systems reveal that the autophagy-lysosome system is the major pathway for Tau degradation in this context. These results demonstrate that UBE4B, a miR-9 target gene, promotes autophagy-mediated Tau degradation together with STUB1, and is thus an innovative therapeutic approach for AD.

DOI: https://doi.org/10.1038/s41467-021-23597-9