bims-proteo 2020-09-06 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2020–09–06
35 papers selected by
Eric Chevet, INSERM

Inhibition of proteasome reveals basal mitochondrial ubiquitination.
C-terminal tail length guides insertion and assembly of membrane proteins.
Thioredoxin-Related Transmembrane Proteins: TMX1 and Little Brothers TMX2, TMX3, TMX4 and TMX5.
Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway.
Small molecule strategies to harness the unfolded protein response: where do we go from here?
Elevated post-ischemic ubiquitination results from suppression of deubiquitinase activity and not proteasome inhibition.
The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation.
Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases.
A metabolite binding protein moonlights as a bile-responsive chaperone.
Ubiquitination of TLR3 by TRIM3 signals its ESCRT-mediated trafficking to the endolysosomes for innate antiviral response.
Epr1, a UPR-upregulated soluble autophagy receptor for reticulophagy.
HSP70 regulates Eg5 distribution within the mitotic spindle and modulates the cytotoxicity of Eg5 inhibitors.
The Roles of Ubiquitin in Mediating Autophagy.
The SUMO conjugase Ubc9 protects Dopaminergic cells from cytotoxicity and enhances the stability of α-synuclein in Parkinson's disease models.
Contributions of alveolar epithelial cell quality control to pulmonary fibrosis.
The regulation of mitochondrial homeostasis by the Ubiquitin Proteasome System.
Inhibition of ER stress improves progressive motor deficits in a REEP1-null mouse model of hereditary spastic paraplegia.
Endoplasmic reticulum stress causes insulin resistance by inhibiting delivery of newly synthesised insulin receptors to the cell surface.
PDPK1 regulates autophagosome biogenesis by binding to PIK3C3.
The E3 Ubiquitin-Protein Ligase Cullin 3 Regulates HIV-1 Transcription.
The Effect of Dysfunctional Ubiquitin Enzymes in the Pathogenesis of Most Common Diseases.
Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.
Heat Shock Protein 70 (HSP70) Induction: Chaperonotherapy for Neuroprotection after Brain Injury.
Effect of the disulfide isomerase PDIa4 on the antibody production of Chinese hamster ovary cells.
N-glycosylation and ubiquitinylation of PD-L1 do not restrict interaction with BMS-202: A molecular modeling study.
Hsf1 on a leash - controlling the heat shock response by chaperone titration.
Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?
A New Endoplasmic Reticulum (ER)-Targeting Fluorescent Probe for the Imaging of Cysteine in Living Cells.
Targeted modulation of E3 ligases using engineered ubiquitin variants.
Ubiquitin ligase TRUSS augments the expression of interleukin-10 via proteasomal processing of NF-κB1/p105 to NF-κB/p50.
The DNA methyltransferase DNMT3A contributes to autophagy long-term memory.
Hrd1-mediated ACLY Ubiquitination alleviate NAFLD in db/db mice.
Proteostasis Regulators Restore Function of Epilepsy-Associated GABAA Receptors.
Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae.
DNA methylation downregulated ZDHHC1 suppresses tumor growth by altering cellular metabolism and inducing oxidative/ER stress-mediated apoptosis and pyroptosis.

J Proteomics. 2020 Aug 31. pii: S1874-3919(20)30317-1. [Epub ahead of print] 103949

Inhibition of proteasome reveals basal mitochondrial ubiquitination.

Prasad Sulkshane, Inbar Duek, Jonathan Ram, Anita Thakur, Noa Reis, Tamar Ziv, Michael H Glickman.

  Strict quality control for mitochondrial proteins is necessary to ensure cell homeostasis. Two cellular pathways-Ubiquitin Proteasome System (UPS) and autophagy-contribute to mitochondrial homeostasis under stressful conditions. Here, we investigate changes to the mitochondria proteome and to the ubiquitin landscape at mitochondria in response to proteasome inhibition. Treatment of HeLa cells devoid of Parkin, the primary E3 ligase responsible for mitophagy, with proteasome inhibitor MG132 for a few hours caused mitochondrial oxidative stress and fragmentation, reduced energy output, and increased mitochondrial ubiquitination without inducing mitophagy. Overexpression of Parkin did not show any induction of mitophagy in response to MG132 treatment. Analysis of ubiquitin chains on isolated mitochondria revealed predominance of K48, K29 and K63-linked polyubiquitin. Interestingly, of all ubiquitinated mitochondrial proteins detected in response to MG132 treatment, a majority (≥90%) were intramitochondrial irrespective of Parkin expression. However, overall levels of these ubiquitinated mitochondrial proteins did not change significantly upon proteasome inhibition when evaluated by quantitative proteomics (LFQ and SILAC), suggesting that only a small portion are ubiquitinated under basal conditions. Another aspect of proteasome inhibition is significant enrichment of UPS, lysosomal and phagosomal components, and other heat shock proteins associated with isolated mitochondria. Taken together, our study highlights a critical role of UPS for ubiquitinating and removing imported proteins as part of a basal mitochondrial quality control system independent of Parkin. SIGNIFICANCE: As centers of cellular bioenergetics, numerous metabolic pathways and signaling cascades, the health of mitochondria is of utmost importance for ensuring cell survival. Due to their unique physiology, mitochondria are constantly subjected to damaging oxidative radicals (ROS) and protein import-related stress due to buildup of unfolded aggregate-prone proteins. Thus, for quality control purposes, mitochondria are constantly under surveillance by Autophagy and the Ubiquitin Proteasome System (UPS), both of which share ubiquitin as a common signal. The ubiquitin landscape of mitochondria has been studied in detail under stressful conditions, however, little is known about basal mitochondrial ubiquitination. Our study reveals that the extent of ubiquitination at mitochondria greatly increases upon proteasome inhibition, pointing to a large number of potential substrates for proteasomal degradation. Interestingly, most of the ubiquitination occurs on intramitochondrial proteins, components of the electron transport chain (ETC) and matrix-resident metabolic enzymes in particular. Moreover, numerous cytosolic UPS components, chaperones and autophagy-lysosomal proteins were recruited to mitochondria upon proteasome inhibition. Taken together, this suggests that the levels and functions of mitochondrial proteins are constantly regulated through ubiquitin-dependent proteasomal degradation even under basal conditions. Unclogging mitochondrial import channels may provide a mechanism to alleviate stress associated with mitochondrial protein import or to adapt cells according to their metabolic needs. Therefore, targeting the mitochondrial ubiquitination/deubiquitination machinery, such as improving the therapeutic potency of proteasome inhibitors, may provide an additional therapeutic arsenal against tumors.

Keywords:  Mitochondria; Mitostasis; Proteasome; Quantitative proteomics; Ubiquitin

DOI:  https://doi.org/10.1016/j.jprot.2020.103949
J Biol Chem. 2020 Sep 02. pii: jbc.RA120.012992. [Epub ahead of print]

C-terminal tail length guides insertion and assembly of membrane proteins.

Sha Sun, Malaiyalam Mariappam.

  A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multi-protein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ~60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ~80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.

Keywords:  endoplasmic reticulum (ER); membrane protein; protein assembly; protein degradation; translocation

DOI:  https://doi.org/10.1074/jbc.RA120.012992
Cells. 2020 Aug 31. pii: E2000. [Epub ahead of print]9(9):

Thioredoxin-Related Transmembrane Proteins: TMX1 and Little Brothers TMX2, TMX3, TMX4 and TMX5.

Concetta Guerra, Maurizio Molinari.

  The endoplasmic reticulum (ER) is site of synthesis and maturation of membrane and secretory proteins in eukaryotic cells. The ER contains more than 20 members of the Protein Disulfide Isomerase (PDI) family. These enzymes regulate formation, isomerization and disassembly of covalent bonds between cysteine residues. As such, PDIs ensure protein folding, which is required to attain functional and transport-competent structure, and protein unfolding, which facilitates dislocation of defective gene products across the ER membrane for ER-associated degradation (ERAD). The PDI family includes over a dozen of soluble members and few membrane-bound ones. Among these latter, there are five PDIs grouped in the thioredoxin-related transmembrane (TMX) protein family. In this review, we summarize the current knowledge on TMX1, TMX2, TMX3, TMX4 and TMX5, their structural features, regulation and roles in biogenesis and control of the mammalian cell's proteome.

Keywords:  ERAD; PDI; TMX; endoplasmic reticulum; folding

DOI:  https://doi.org/10.3390/cells9092000
Cells. 2020 Aug 29. pii: E1994. [Epub ahead of print]9(9):

Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway.

Philip J Robinson, Neil J Bulleid.

  Disulphide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulphide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulphide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulphide bond formation is reviewed with emphasis on events that occur during ER entry. Disulphide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulphide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulphide bond formation and identifies key questions that need to be addressed to further understand this fundamental process.

Keywords:  ER; PDI; disulphide formation; protein folding; protein secretion; protein synthesis

DOI:  https://doi.org/10.3390/cells9091994
J Biol Chem. 2020 Sep 04. pii: jbc.REV120.010218. [Epub ahead of print]

Small molecule strategies to harness the unfolded protein response: where do we go from here?

Julia M D Grandjean, R Luke Wiseman.

  The unfolded protein response (UPR) plays a central role in regulating endoplasmic reticulum (ER) and global cellular physiology in response to pathologic ER stress. The UPR is comprised of three signaling pathways activated downstream of the ER membrane proteins IRE1, ATF6, and PERK. Once activated, these proteins initiate transcriptional and translational signaling that functions to alleviate ER stress, adapt cellular physiology, and dictate cell fate. Imbalances in UPR signaling are implicated in the pathogenesis of numerous, etiologically-diverse diseases including many neurodegenerative diseases, protein misfolding diseases, diabetes, ischemic disorders, and cancer. This has led to significant interest in establishing pharmacologic strategies to selectively modulate IRE1, ATF6, or PERK signaling to both ameliorate pathologic imbalances in UPR signaling implicated in these different diseases, and to define the importance of the UPR in diverse cellular and organismal contexts. Recently, there has been significant progress in the identification and characterization of UPR modulating compounds, providing new opportunities to probe the pathologic and potentially therapeutic implications of UPR signaling in human disease. Here, we describe currently available UPR modulating compounds, specifically highlighting the strategies used for their discovery and specific advantages and disadvantages in their application for probing UPR function. Furthermore, we discuss lessons learned from the application of these compounds in cellular and in vivo models to identify favorable compound properties that can help drive the further translational development of selective UPR modulators for human disease.

Keywords:  endoplasmic reticulum stress (ER stress); proteostasis; small molecule; stress response; unfolded protein response (UPR)

DOI:  https://doi.org/10.1074/jbc.REV120.010218
Cell Mol Life Sci. 2020 Sep 05.

Elevated post-ischemic ubiquitination results from suppression of deubiquitinase activity and not proteasome inhibition.

Timo Kahles, Carrie Poon, Liping Qian, Victoria Palfini, Shanmukha Priya Srinivasan, Shilpa Swaminathan, Ismary Blanco, Reunet Rodney-Sandy, Costantino Iadecola, Ping Zhou, Karin Hochrainer.

  Cerebral ischemia-reperfusion increases intraneuronal levels of ubiquitinated proteins, but the factors driving ubiquitination and whether it results from altered proteostasis remain unclear. To address these questions, we used in vivo and in vitro models of cerebral ischemia-reperfusion, in which hippocampal slices were transiently deprived of oxygen and glucose to simulate ischemia followed by reperfusion, or the middle cerebral artery was temporarily occluded in mice. We found that post-ischemic ubiquitination results from two key steps: restoration of ATP at reperfusion, which allows initiation of protein ubiquitination, and free radical production, which, in the presence of sufficient ATP, increases ubiquitination above pre-ischemic levels. Surprisingly, free radicals did not augment ubiquitination through inhibition of the proteasome as previously believed. Although reduced proteasomal activity was detected after ischemia, this was neither caused by free radicals nor sufficient in magnitude to induce appreciable accumulation of proteasomal target proteins or ubiquitin-proteasome reporters. Instead, we found that ischemia-derived free radicals inhibit deubiquitinases, a class of proteases that cleaves ubiquitin chains from proteins, which was sufficient to elevate ubiquitination after ischemia. Our data provide evidence that free radical-dependent deubiquitinase inactivation rather than proteasomal inhibition drives ubiquitination following ischemia-reperfusion, and as such call for a reevaluation of the mechanisms of post-ischemic ubiquitination, previously attributed to altered proteostasis. Since deubiquitinase inhibition is considered an endogenous neuroprotective mechanism to shield proteins from oxidative damage, modulation of deubiquitinase activity may be of therapeutic value to maintain protein integrity after an ischemic insult.

Keywords:  Cerebral ischemia–reperfusion; Deubiquitinase inhibition; Free radical production; Ubiquitin

DOI:  https://doi.org/10.1007/s00018-020-03625-5
Mol Cell Proteomics. 2020 Aug 31. pii: mcp.RA120.002050. [Epub ahead of print]

The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation.

Teresa M Buck, Xuemei Zeng, Pamela S Cantrell, Richard T Cattley, Zikri Hasanbasri, Megan E Yates, Diep Nguyen, Nathan A Yates, Jeffrey L Brodsky.

  Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin-proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques-including mass spectrometry (MS)-have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem mass spectrometry. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation was dependent on ERAD-associated ubiquitin ligases and regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

Keywords:  ERAD; Mass Spectrometry; Protein Degradation*; Protein Folding*; Protein complex analysis; Ubiquitin; Yeast*; proteasome

DOI:  https://doi.org/10.1074/mcp.RA120.002050
Biochemistry. 2020 Aug 31.

Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases.

Chad S Hewitt, Aaron D Krabill, Chittaranjan Das, Daniel P Flaherty.

Ubiquitin (Ub) is a small highly conserved protein that is covalently attached to substrate proteins as a post translational modification to regulate numerous signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. The role of Ub as a post-translational signal in these pathways results in a highly regulated homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUB) in healthy eukaryotic systems. One such DUB known as Ubiquitin C-terminal Hydrolase L1 (UCHL1) is endogenously expressed in the central nervous system under normal physiological conditions but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. There is a significant lack of UCHL1 probing strategies and development of a selective Ub-variant (UbV) to probe UCHL1's role in these disease states would be immensely beneficial. To this end, we describe a rational design approach to investigate Ub mutants that lend selectivity to UCHL1 over the close structural homolog UCHL3 and other DUB families. UbT9F/T66K displayed appreciable selectivity for recombinantly expressed UCHL1 over UCHL3 compared to WT-Ub in in vitro assays. By appending a reactive electrophile to the C-terminus of the Ub mutant we created the first activity-based probe (ABP) with demonstrated selectivity for UCH family DUBs over other families in cell lysates. Further analysis of kinetics of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into future design toward UCHL1-selective Ub-ABP probe for applications in the cellular environment. These studies serve as a proof-of-concept of the viability of in silico rational design of ubiquitin variants for UCH family DUBs as a step toward the development macromolecular UCHL1 inhibitors.

DOI: https://doi.org/10.1021/acs.biochem.9b01076
EMBO J. 2020 Sep 03. e104231

A metabolite binding protein moonlights as a bile-responsive chaperone.

Changhan Lee, Patrick Betschinger, Kevin Wu, Dawid S Żyła, Rudi Glockshuber, James Ca Bardwell.

  Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.

Keywords:  chaperone; protein folding

DOI:  https://doi.org/10.15252/embj.2019104231
Proc Natl Acad Sci U S A. 2020 Sep 02. pii: 202002472. [Epub ahead of print]

Ubiquitination of TLR3 by TRIM3 signals its ESCRT-mediated trafficking to the endolysosomes for innate antiviral response.

Wei-Wei Li, Ying Nie, Yan Yang, Yong Ran, Wei-Wei Luo, Mei-Guang Xiong, Su-Yun Wang, Zhi-Sheng Xu, Yan-Yi Wang.

  Trafficking of toll-like receptor 3 (TLR3) from the endoplasmic reticulum (ER) to endolysosomes and its subsequent proteolytic cleavage are required for it to sense viral double-stranded RNA (dsRNA) and trigger antiviral response, yet the underlying mechanisms remain enigmatic. We show that the E3 ubiquitin ligase TRIM3 is mainly located in the Golgi apparatus and transported to the early endosomes upon stimulation with the dsRNA analog poly(I:C). TRIM3 mediates K63-linked polyubiquitination of TLR3 at K831, which is enhanced following poly(I:C) stimulation. The polyubiquitinated TLR3 is recognized and sorted by the ESCRT (endosomal sorting complex required for transport) complexes to endolysosomes. Deficiency of TRIM3 impairs TLR3 trafficking from the Golgi apparatus to endosomes and its subsequent activation. Trim3 -/- cells and mice express lower levels of antiviral genes and show lower levels of inflammatory response following poly(I:C) but not lipopolysaccharide (LPS) stimulation. These findings suggest that TRIM3-mediated polyubiquitination of TLR3 represents a feedback-positive regulatory mechanism for TLR3-mediated innate immune and inflammatory responses.

Keywords:  ESCRT; TLR3; TRIM3; innate antiviral response; polyubiquitination

DOI:  https://doi.org/10.1073/pnas.2002472117
Autophagy. 2020 Aug 31.

Epr1, a UPR-upregulated soluble autophagy receptor for reticulophagy.

Dan Zhao, Li-Lin Du.

  The endoplasmic reticulum (ER) is a major site of protein folding. Perturbations in the folding capacity of the ER result in ER stress. ER stress triggers autophagic degradation of the ER (reticulophagy). Molecular mechanisms underlying ER stress-induced reticulophagy remain largely unknown. Our recent study identified a soluble protein, Epr1, as an autophagy receptor for ER stress-induced reticulophagy in the fission yeast Schizosaccharomyces pombe. Epr1 can interact simultaneously with Atg8 and a VAP family integral ER membrane protein, and thereby act as a bridging molecule between them. VAP family proteins contribute to reticulophagy by not only connecting Atg8 to the ER membrane through Epr1, but also by supporting the ER-plasma membrane contact. The expression of Epr1 is upregulated during ER stress in a manner dependent on the unfolded protein response (UPR) regulator Ire1. Ire1 promotes reticulophagy by upregulating Epr1.

Keywords:  ER stress; ER-phagy; ER-plasma membrane contact; Ire1; VAP; reticulophagy; selective autophagy; unfolded protein response (UPR)

DOI:  https://doi.org/10.1080/15548627.2020.1816665
Cell Death Dis. 2020 Sep 01. 11(8): 715

HSP70 regulates Eg5 distribution within the mitotic spindle and modulates the cytotoxicity of Eg5 inhibitors.

Chieh-Ting Fang, Hsiao-Hui Kuo, Shao-Chun Hsu, Ling-Huei Yih.

The heat shock protein 70 (HSP70) is a conserved molecular chaperone and proteostasis regulator that protects cells from pharmacological stress and promotes drug resistance in cancer cells. In this study, we found that HSP70 may promote resistance to anticancer drugs that target the mitotic kinesin, Eg5, which is essential for assembly and maintenance of the mitotic spindle and cell proliferation. Our data show that loss of HSP70 activity enhances Eg5 inhibitor-induced cytotoxicity and spindle abnormalities. Furthermore, HSP70 colocalizes with Eg5 in the mitotic spindle, and inhibition of HSP70 disrupts this colocalization. Inhibition or depletion of HSP70 also causes Eg5 to accumulate at the spindle pole, altering microtubule dynamics and leading to chromosome misalignment. Using ground state depletion microscopy followed by individual molecule return (GSDIM), we found that HSP70 inhibition reduces the size of Eg5 ensembles and prevents their localization to the inter-polar region of the spindle. In addition, bis(maleimido)hexane-mediated protein-protein crosslinking and proximity ligation assays revealed that HSP70 inhibition deregulates the interaction between Eg5 tetramers and TPX2 at the spindle pole, leading to their accumulation in high-molecular-weight complexes. Finally, we showed that the passive substrate-binding activity of HSP70 is required for appropriate Eg5 distribution and function. Together, our results show that HSP70 substrate-binding activity may regulate proper assembly of Eg5 ensembles and Eg5-TPX2 complexes to modulate mitotic distribution/function of Eg5. Thus, HSP70 inhibition may sensitize cancer cells to Eg5 inhibitor-induced cytotoxicity.

DOI: https://doi.org/10.1038/s41419-020-02919-7
Cells. 2020 Sep 02. pii: E2025. [Epub ahead of print]9(9):

The Roles of Ubiquitin in Mediating Autophagy.

Zhangyuan Yin, Hana Popelka, Yuchen Lei, Ying Yang, Daniel J Klionsky.

  Ubiquitination, the post-translational modification essential for various intracellular processes, is implicated in multiple aspects of autophagy, the major lysosome/vacuole-dependent degradation pathway. The autophagy machinery adopted the structural architecture of ubiquitin and employs two ubiquitin-like protein conjugation systems for autophagosome biogenesis. Ubiquitin chains that are attached as labels to protein aggregates or subcellular organelles confer selectivity, allowing autophagy receptors to simultaneously bind ubiquitinated cargos and autophagy-specific ubiquitin-like modifiers (Atg8-family proteins). Moreover, there is tremendous crosstalk between autophagy and the ubiquitin-proteasome system. Ubiquitination of autophagy-related proteins or regulatory components plays significant roles in the precise control of the autophagy pathway. In this review, we summarize and discuss the molecular mechanisms and functions of ubiquitin and ubiquitination, in the process and regulation of autophagy.

Keywords:  autophagy; lysosome; selective autophagy; ubiquitin; ubiquitination

DOI:  https://doi.org/10.3390/cells9092025
eNeuro. 2020 Sep 04. pii: ENEURO.0134-20.2020. [Epub ahead of print]

The SUMO conjugase Ubc9 protects Dopaminergic cells from cytotoxicity and enhances the stability of α-synuclein in Parkinson's disease models.

Dinesh Kumar Verma, Anurupa Ghosh, Lindsey Ruggiero, Etienne Cartier, Eric Janezic, Dionne Williams, Eui-Gil Jung, Michael Moore, Jong Bok Seo, Yong-Hwan Kim.

  Small Ubiquitin-like Modifier (SUMO) is a widespread regulatory mechanism of post-translational modification that induces rapid and reversible changes in protein function and stability. Using SUMO conjugase Ubc9 overexpressing or knock-down cells in Parkinson's disease (PD) models, we demonstrate that SUMOylation protects dopaminergic cells against MPP+ or preformed fibrils (PFF) of alpha-synuclein induced toxicities in cell viability and cytotoxicity assays. In the mechanism of protection, Ubc9 overexpression significantly suppressed the MPP+ or PFF-induced ROS generation, while Ubc9-RNAi enhanced the toxicity-induced ROS production. Further, PFF-mediated protein aggregation was exacerbated by Ubc9-RNAi in Thioflavin T staining, compared to NC1 controls. In cycloheximide-based protein stability assays, higher protein level of α-synuclein was identified in Ubc9-EGFP than in EGFP cells. Since there was no difference in endogenous mRNA levels of α-synuclein between Ubc9 and EGFP cells in qRT-PCR, we assessed the mechanisms of SUMO-mediated delayed α-synuclein degradation via MG132, proteasomal inhibitor and PMA, lysosomal degradation inducer. Ubc9-mediated SUMOylated α-synuclein avoided PMA-induced lysosomal degradation due to its high solubility. Our results suggest that Ubc9 enhances the levels of SUMO1 and Ubiquitin on α-synuclein and interrupts SUMO1 removal from α-synuclein. In immunohistochemistry, dopaminergic axon tips in the striatum and cell bodies in the Substantia Nigra from Ubc9-overexpressing transgenic mice were protected from MPTP toxicities compared to WT siblings. Our results support that SUMOylation can be a regulatory target to protect dopaminergic neurons from oxidative stress and protein aggregation, with the implication that high levels of SUMOylation in dopaminergic neurons can prevent the pathological progression of PD.Significance Statement We tested if SUMOylation enhances the solubility of aggregation-prone proteins such as α-synuclein to prevent protein aggregation induced by oxidative stress and/or preformed fibrils (PFF) of α-synuclein. Here, we demonstrate that high levels of SUMOylation mediated by Ubc9 overexpression protect dopaminergic cells from MPTP- (MPP+) or PFF-induced toxicities. The protective effects are derived from the inhibition of ROS generation and protein aggregation. Interestingly, SUMOylated α-synuclein avoided lysosomal degradation, which was not detrimental. Ubiquitin binding to lysine residues may not compete with SUMO binding to determine the protein half-life of α-synuclein. Our findings strongly suggest that the regulation of SUMO conjugation to α-synuclein can be a novel therapeutic target to prevent the formation of Lewy bodies and ROS generation.

Keywords:  SUMOylation; Ubc9; alpha-Synuclein; degradation; lysosome; proteostasis

DOI:  https://doi.org/10.1523/ENEURO.0134-20.2020
J Clin Invest. 2020 Sep 01. pii: 139519. [Epub ahead of print]

Contributions of alveolar epithelial cell quality control to pulmonary fibrosis.

Jeremy Katzen, Michael F Beers.

Epithelial cell dysfunction has emerged as a central component in the pathophysiology of diffuse parenchymal diseases including idiopathic pulmonary fibrosis (IPF). Alveolar type 2 (AT2) cells represent a metabolically active lung cell population important for surfactant biosynthesis and alveolar homeostasis. AT2 cells and other distal lung epithelia, like all eukaryotic cells, contain an elegant quality control (QC) network to respond to intrinsic metabolic and biosynthetic challenges imparted by mutant protein conformers, dysfunctional subcellular organelles, and dysregulated telomeres. Failed AT2 QC components (ubiquitin-proteasome system, unfolded protein response, macroautophagy, mitophagy, and telomere maintenance) result in diverse cellular endophenotypes and molecular signatures including ER stress, defective autophagy, mitochondrial dysfunction, apoptosis, inflammatory cell recruitment, profibrotic signaling, and altered progenitor function that ultimately converge to drive downstream fibrotic remodeling in the IPF lung. As this complex network becomes increasingly better understood, opportunities will emerge to identify targets and therapeutic strategies for IPF.

DOI: https://doi.org/10.1172/JCI139519
Biochim Biophys Acta Bioenerg. 2020 Aug 27. pii: S0005-2728(20)30152-3. [Epub ahead of print] 148302

The regulation of mitochondrial homeostasis by the Ubiquitin Proteasome System.

Cynthia Alsayyah, Oznur Ozturk, Laetitia Cavellini, Naïma Belgareh-Touzé, Mickael M Cohen.

  From mitochondrial quality control pathways to the regulation of specific functions, the Ubiquitin Proteasome System (UPS) could be compared to a Swiss knife without which mitochondria could not maintain its integrity in the cell. Here, we review the mechanisms that the UPS employs to regulate mitochondrial function and efficiency. For this purpose, we depict how Ubiquitin and the Proteasome participate in diverse quality control pathways that safeguard entry into the mitochondrial compartment. A focus is then achieved on the UPS-mediated control of the yeast mitofusin Fzo1 which provides insights into the complex regulation of this particular protein in mitochondrial fusion. We ultimately dissect the mechanisms by which the UPS controls the degradation of mitochondria by autophagy in both mammalian and yeast systems. This organization should offer a useful overview of this abundant but fascinating literature on the crosstalks between mitochondria and the UPS.

Keywords:  Mitochondria; Mitochondrial Quality Control; Mitochondrial fusion; Mitophagy; Proteasome; Ubiquitin

DOI:  https://doi.org/10.1016/j.bbabio.2020.148302
Biol Open. 2020 Sep 02. pii: bio.054296. [Epub ahead of print]

Inhibition of ER stress improves progressive motor deficits in a REEP1-null mouse model of hereditary spastic paraplegia.

Bingjie Wang, You Yu, Lai Wei, Yan Zhang.

  Hereditary spastic paraplegias (HSPs) are genetic neurodegenerative diseases. HSPs are characterized by lower-extremity weakness and spasticity. However, there is no specific clinical treatment strategy to prevent or reverse nerve degeneration in HSPs. Mutations in receptor expression-enhancing protein 1 (REEP1) are well-recognized and relatively common causes of autosomal dominant HSPs. REEP1 modifies the endoplasmic reticulum (ER) shape, and is implicated in the ER stress response. Defects in the ER stress response seem to be crucial mechanisms underlying HSP neurodegeneration. Here, we report that REEP1-/- mice exhibit progressive motor deficits, along with denervation of neuromuscular junctions and increased ER stress. Moreover, marked axonal degeneration and morphological abnormalities are observed. In this study, we treated both REEP1-/- and wild-type (WT) mice with salubrinal, which is a specific inhibitor of ER stress, and we observed increased nerve-muscle connections and enhanced motor functions. Our data highlight the importance of ER homeostasis in HSPs, providing new opportunities for HSP treatment.

Keywords:  Endoplasmic reticulum stress; Hereditary spastic paraplegias; Receptor expression-enhancing protein 1; Salubrinal

DOI:  https://doi.org/10.1242/bio.054296
Mol Biol Cell. 2020 Sep 02. mbcE18010013

Endoplasmic reticulum stress causes insulin resistance by inhibiting delivery of newly synthesised insulin receptors to the cell surface.

Max Brown, Samantha Dainty, Natalie Strudwick, Adina D Mihai, Jamie N Watson, Robina Dendooven, Adrienne W Paton, James C Paton, Martin Schröder.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signalling network known as the unfolded protein response (UPR). Here we characterise how ER stress and the UPR inhibit insulin signalling. We find that ER stress inhibits insulin signalling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesised insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signalling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible FV2E dimerisation domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signalling events in the UPR, such as activation of the JNK MAP kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signalling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesised insulin receptors in ER-stressed cells.

DOI: https://doi.org/10.1091/mbc.E18-01-0013
Autophagy. 2020 Sep 02.

PDPK1 regulates autophagosome biogenesis by binding to PIK3C3.

Boli Hu, Yina Zhang, Tingjuan Deng, Jinyan Gu, Juan Liu, Hui Yang, Yuting Xu, Yan Yan, Fan Yang, Heng Zhang, Yulan Jin, Jiyong Zhou.

  PDPK1 (3-phosphoinositide dependent protein kinase 1) is a phosphorylation-regulated kinase that plays a central role in activating multiple signaling pathways and cellular processes. Here, this study shows that PDPK1 turns on macroautophagy/autophagy as a SUMOylation-regulated kinase. In vivo data demonstrate that the SUMO modification of PDPK1 is a physiological feature in the brain and that it can be induced by viral infections. The SUMOylated PDPK1 regulates its own phosphorylation and subsequent activation of the AKT1 (AKT serine/threonine kinase 1)-MTOR (mechanistic target of rapamycin kinase) pathway. However, SUMOylation of PDPK1 is inhibited by binding to PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3). The nonSUMOylated PDPK1 then tethers LC3 to the endoplasmic reticulum to initiate autophagy, and it acts as a key component in forming the autophagic vacuole. Collectively, this study reveals the intricate molecular regulation of PDPK1 by post-translational modification in controlling autophagosome biogenesis, and it highlights the role of PDPK1 as a sensor of cellular stress and regulator of autophagosome biogenesis.

Keywords:  AKT1-MTOR; PDPK1; PIK3C3; SUMOylation; autophagy

DOI:  https://doi.org/10.1080/15548627.2020.1817279
Cells. 2020 Sep 01. pii: E2010. [Epub ahead of print]9(9):

The E3 Ubiquitin-Protein Ligase Cullin 3 Regulates HIV-1 Transcription.

Simon Langer, Xin Yin, Arturo Diaz, Alex J Portillo, David E Gordon, Umu H Rogers, John M Marlett, Nevan J Krogan, John A T Young, Lars Pache, Sumit K Chanda.

  The infectious life cycle of the human immunodeficiency virus type 1 (HIV-1) is characterized by an ongoing battle between a compendium of cellular proteins that either promote or oppose viral replication. On the one hand, HIV-1 utilizes dependency factors to support and sustain infection and complete the viral life cycle. On the other hand, both inducible and constitutively expressed host factors mediate efficient and functionally diverse antiviral processes that counteract an infection. To shed light into the complex interplay between HIV-1 and cellular proteins, we previously performed a targeted siRNA screen to identify and characterize novel regulators of viral replication and identified Cullin 3 (Cul3) as a previously undescribed factor that negatively regulates HIV-1 replication. Cul3 is a component of E3-ubiquitin ligase complexes that target substrates for ubiquitin-dependent proteasomal degradation. In the present study, we show that Cul3 is expressed in HIV-1 target cells, such as CD4+ T cells, monocytes, and macrophages and depletion of Cul3 using siRNA or CRISPR/Cas9 increases HIV-1 infection in immortalized cells and primary CD4+ T cells. Conversely, overexpression of Cul3 reduces HIV-1 infection in single replication cycle assays. Importantly, the antiviral effect of Cul3 was mapped to the transcriptional stage of the viral life cycle, an effect which is independent of its role in regulating the G1/S cell cycle transition. Using isogenic viruses that only differ in their promotor region, we find that the NF-κB/NFAT transcription factor binding sites in the LTR are essential for Cul3-dependent regulation of viral gene expression. Although Cul3 effectively suppresses viral gene expression, HIV-1 does not appear to antagonize the antiviral function of Cul3 by targeting it for degradation. Taken together, these results indicate that Cul3 is a negative regulator of HIV-1 transcription which governs productive viral replication in infected cells.

Keywords:  Cullin 3; HIV-1; NF-κB signaling; ubiquitin protein ligase; viral gene transcription

DOI:  https://doi.org/10.3390/cells9092010
Int J Mol Sci. 2020 Sep 01. pii: E6335. [Epub ahead of print]21(17):

The Effect of Dysfunctional Ubiquitin Enzymes in the Pathogenesis of Most Common Diseases.

Gizem Celebi, Hale Kesim, Ebru Ozer, Ozlem Kutlu.

  Ubiquitination is a multi-step enzymatic process that involves the marking of a substrate protein by bonding a ubiquitin and protein for proteolytic degradation mainly via the ubiquitin-proteasome system (UPS). The process is regulated by three main types of enzymes, namely ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Under physiological conditions, ubiquitination is highly reversible reaction, and deubiquitinases or deubiquitinating enzymes (DUBs) can reverse the effect of E3 ligases by the removal of ubiquitin from substrate proteins, thus maintaining the protein quality control and homeostasis in the cell. The dysfunction or dysregulation of these multi-step reactions is closely related to pathogenic conditions; therefore, understanding the role of ubiquitination in diseases is highly valuable for therapeutic approaches. In this review, we first provide an overview of the molecular mechanism of ubiquitination and UPS; then, we attempt to summarize the most common diseases affecting the dysfunction or dysregulation of these mechanisms.

Keywords:  DUBs; E3s; UPS; cancer; immune-related diseases; neurodegenerative disease; ubiquitination

DOI:  https://doi.org/10.3390/ijms21176335
Science. 2020 Sep 04. pii: eaaz7714. [Epub ahead of print]369(6508):

Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.

Justyna Sawa-Makarska, Verena Baumann, Nicolas Coudevylle, Sören von Bülow, Veronika Nogellova, Christine Abert, Martina Schuschnig, Martin Graef, Gerhard Hummer, Sascha Martens.

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.

DOI: https://doi.org/10.1126/science.aaz7714
Cells. 2020 Sep 02. pii: E2020. [Epub ahead of print]9(9):

Heat Shock Protein 70 (HSP70) Induction: Chaperonotherapy for Neuroprotection after Brain Injury.

Jong Youl Kim, Sumit Barua, Mei Ying Huang, Joohyun Park, Midori A Yenari, Jong Eun Lee.

  The 70 kDa heat shock protein (HSP70) is a stress-inducible protein that has been shown to protect the brain from various nervous system injuries. It allows cells to withstand potentially lethal insults through its chaperone functions. Its chaperone properties can assist in protein folding and prevent protein aggregation following several of these insults. Although its neuroprotective properties have been largely attributed to its chaperone functions, HSP70 may interact directly with proteins involved in cell death and inflammatory pathways following injury. Through the use of mutant animal models, gene transfer, or heat stress, a number of studies have now reported positive outcomes of HSP70 induction. However, these approaches are not practical for clinical translation. Thus, pharmaceutical compounds that can induce HSP70, mostly by inhibiting HSP90, have been investigated as potential therapies to mitigate neurological disease and lead to neuroprotection. This review summarizes the neuroprotective mechanisms of HSP70 and discusses potential ways in which this endogenous therapeutic molecule could be practically induced by pharmacological means to ultimately improve neurological outcomes in acute neurological disease.

Keywords:  brain injury; chaperone neuroprotection; heat shock protein 70; pharmacological induction

DOI:  https://doi.org/10.3390/cells9092020
J Biosci Bioeng. 2020 Aug 30. pii: S1389-1723(20)30300-5. [Epub ahead of print]

Effect of the disulfide isomerase PDIa4 on the antibody production of Chinese hamster ovary cells.

Kei Komatsu, Kento Kumon, Mayuno Arita, Masayoshi Onitsuka, Takeshi Omasa, Masafumi Yohda.

  Therapeutic monoclonal antibodies recognize and bind specific molecules on the surface of target cells, stimulating the immune system, which can attack these targeted cells. These antibodies are produced by mammalian cells, including Chinese hamster ovary (CHO) cells, because the formation of antibodies requires complicated posttranslational modifications, including peptidyl-prolyl cis/trans isomerization, disulfide bond formation, and glycosylation. Currently, it is thought that the efficient production of secretory proteins is limited by posttranslational processes. The ER is the biosynthesis site of all secreted and membrane proteins. The accumulation of unfolded proteins in the ER causes the ER stress response. During the ER stress state, various molecular chaperones are expressed to prevent proteins from the aggregate formation. The molecular chaperone involved in ER stress likely plays an essential role in the production of secretory proteins. The purpose of this study was to improve the production of monoclonal antibodies by cells. We elucidated the function of ER chaperones in the production of a monoclonal antibody. First, we quantitatively measured the mRNA expression levels of protein disulfide-isomerase family members. In CHO HcD6 cells treated with tunicamycin, the expression level of pdia4 was significantly increased. Second, we investigated the relationship between PDIa4 and antibody productivity in pdia4-knockdown cells. Both a decrease in the amount of secreted antibody and the accumulation of immature antibodies inside the cells were observed. Recombinant PDIa4 was able to refold the antibodies and Fabs. These results indicate that PDIa4 affects the production of monoclonal antibodies by catalyzing disulfide bond formation in these antibodies in CHO cells.

Keywords:  Antibody; Chaperone; Chinese hamster ovary; Folding; PDIa4; Protein disulfide-isomerase

DOI:  https://doi.org/10.1016/j.jbiosc.2020.08.001
Comput Biol Chem. 2020 Aug 19. pii: S1476-9271(20)30941-5. [Epub ahead of print]88 107362

N-glycosylation and ubiquitinylation of PD-L1 do not restrict interaction with BMS-202: A molecular modeling study.

Christian Bailly, Gérard Vergoten.

  The Programmed cell Death protein-1/Ligand 1 (PD-1/L1) checkpoint is a major target in oncology. Monoclonal antibodies targeting PD-1 or PD-L1 are used to treat different types of solid tumors and lymphoma. PD-L1-binding small molecules are also actively searched. The lead compound is the biphenyl drug BMS-202 which stabilizes PD-L1 protein dimers and displays a potent antitumor activity in experimental models. Here we have investigated the effect of N-glycosylation (at N35, N192, N200 and N219) and mono-ubiquitination (at K178) of PD-L1 on the interaction with BMS-202 by molecular modeling. Two complementary tridimensional models of PD-L1, based on available crystallographic structures, were constructed with BMS-202 bound. The structures were glycosylated, with a fucosylated bi-antennary N-glycan and ubiquitinated. Model 1 refers to glycoPD-L1 bearing 16 N-glycans, with or without 4 ubiquitin residues. Model 2 presents 8 N-glycans and 2 ubiquitin residues. In both cases, BMS-202 was bound to the protein interface, stabilizing a PD-L1 dimer. The incorporation of the N-glycans or the ubiquitins did not significantly alter the drug-protein recognition. The interface of the drug-stabilized protein dimer is unaffected by the glycosylation or ubiquitination. Calculations of the binding energies indicated that the glycosylation slightly reduces the stability of the drug-protein complexes but does not prevent the drug binding process. Our modeling study suggests that the drug can target efficiently the different forms of PD-L1 in cells, glycosylated, ubiquitinated or not. These models of N-glycosylated and ubiquitinated PD-L1 will be useful to study other PD-L1 protein complexes.

Keywords:  BMS-202; Drug-protein binding; Molecular modelling; PD-L1; Protein glycosylation; Ubiquitin

DOI:  https://doi.org/10.1016/j.compbiolchem.2020.107362
Exp Cell Res. 2020 Aug 27. pii: S0014-4827(20)30495-X. [Epub ahead of print]396(1): 112246

Hsf1 on a leash - controlling the heat shock response by chaperone titration.

Anna E Masser, Michela Ciccarelli, Claes Andréasson.

  Heat shock factor 1 (Hsf1) is an ancient transcription factor that monitors protein homeostasis (proteostasis) and counteracts disturbances by triggering a transcriptional programme known as the heat shock response (HSR). The HSR is transiently activated and upregulates the expression of core proteostasis genes, including chaperones. Dysregulation of Hsf1 and its target genes are associated with disease; cancer cells rely on a constitutively active Hsf1 to promote rapid growth and malignancy, whereas Hsf1 hypoactivation in neurodegenerative disorders results in formation of toxic aggregates. These central but opposing roles highlight the importance of understanding the underlying molecular mechanisms that control Hsf1 activity. According to current understanding, Hsf1 is maintained latent by chaperone interactions but proteostasis perturbations titrate chaperone availability as a result of chaperone sequestration by misfolded proteins. Liberated and activated Hsf1 triggers a negative feedback loop by inducing the expression of key chaperones. Until recently, Hsp90 has been highlighted as the central negative regulator of Hsf1 activity. In this review, we focus on recent advances regarding how the Hsp70 chaperone controls Hsf1 activity and in addition summarise several additional layers of activity control.

Keywords:  Heat shock factor 1; Heat shock response; Hsp70; Hsp90; Misfolded proteins; Protein homeostasis

DOI:  https://doi.org/10.1016/j.yexcr.2020.112246
Bioessays. 2020 Sep 03. e2000036

Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?

Thuy P Dao, Carlos A Castañeda.

  Liquid-liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin-binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.

Keywords:  autophagy; biomolecular condensates; liquid-liquid phase separation; polyubiquitin; proteasomal degradation; protein quality control; ubiquitin shuttle proteins

DOI:  https://doi.org/10.1002/bies.202000036
J Fluoresc. 2020 Sep 01.

A New Endoplasmic Reticulum (ER)-Targeting Fluorescent Probe for the Imaging of Cysteine in Living Cells.

Lei Zhou, Yunxia Li, Aiqin Zhou, Guanghui Zhang, Zhi-Qiang Cheng, Yong-Xi Ge, Shan-Kui Liu, Ricardo Bentes Azevedo, Juan Zhang, Shenglin Jiang, Cheng-Shi Jiang.

  Cysteine (Cys) is an important endogenous amino acid and plays critical physiological roles in living systems. Herein, an endoplasmic reticulum (ER)-targeting fluorescent probe (FER-Cys) was designed and prepared for imaging of Cys in living cells. The probe FER-Cys consists of a fluorescein framework as the fluorescent platform, acrylate group as the response site for the selective recognition of Cys, and ER-specific p-toluenesulfonamide fragment. After the response of probe FER-Cys to Cys, a turn-on fluorescence signal at 546 nm could be detected obviously. The probe FER-Cys further shows desirable selectivity to Cys. Finally, the probe FER-Cys was proven to selectively detect Cys in live cells and successfully image the changes of Cys level in the cell models of H2O2-induced redox imbalance.

Keywords:  Endoplasmic reticulum-targeting; Fluorescent probe; Imaging of cysteine; Redox imbalance; Selective recognition

DOI:  https://doi.org/10.1007/s10895-020-02615-x
FEBS J. 2020 Aug 31.

Targeted modulation of E3 ligases using engineered ubiquitin variants.

Nicole LeBlanc, Evan Mallette, Wei Zhang.

  Ubiquitination plays an essential role in signal transduction to regulate most if not all cellular processes. Among the enzymes that are involved in the ubiquitin (Ub) signaling cascade, tremendous efforts have been focused on elucidating the roles of E3 Ub ligases as they determine the complexity and specificity of ubiquitination. Not surprisingly, the malfunction of E3 ligases is directly implicated in many human diseases, including cancer. Therefore, there is an urgent need to develop potent and specific molecules to modulate E3 ligase activity as intracellular probes for target validation and as pharmacological agents in pre-clinical research. Unfortunately, the progress has been hampered by the dynamic regulation mechanisms for different types of E3 ligases. Here, we summarize the progress of using protein engineering to develop Ub variant (UbV) inhibitors for all major families of E3 ligases and UbV activators for HECT E3s and homodimeric RING E3s. We believe that this provides a general strategy and a valuable toolkit for the research community to inhibit or activate E3 ligases and these synthetic molecules have important implications in exploring protein degradation for drug discovery.

Keywords:  Activator; Cancer; E3 ligases; Inhibitor; Phage display; Protein engineering; Signal transduction; Ubiquitin variant; Ubiquitination

DOI:  https://doi.org/10.1111/febs.15536
Cell Signal. 2020 Sep 01. pii: S0898-6568(20)30243-6. [Epub ahead of print] 109766

Ubiquitin ligase TRUSS augments the expression of interleukin-10 via proteasomal processing of NF-κB1/p105 to NF-κB/p50.

Azfar Jamal, Atahar Husein, Chhagan Bihari, Vijay Kumar.

  The NF-κB/Rel family of transcription factors that play critical roles in a variety of cellular processes. Their production in the cell and physiological activation are tightly regulated. The proteasomal processing of inactive NF-κB1/p105 to active p50, with an anti-inflammatory role, is not well characterized. Here we show that ubiquitin ligase TRUSS is a mediator of transcriptional activation of anti-inflammatory cytokine IL-10 gene. Enforced expression of TRUSS led to enhanced IL-10 expression that could be inhibited in the presence of chemical inhibitors of NF-κB [BAY11-7082] and PI3K/Akt [LY249002] or after p65 overexpression. p50 was actively recruited on IL10 promoter in the presence of TRUSS but competed by p65 for binding. TRUSS facilitated the ubiquitination of NF-κB1/p105 and promoted its proteolytic processing to generate excess of p50. Our immune-histochemical studies confirmed enhanced expression of p105/p50 in the human HCC tumors. Further, the hepatic tumors of HCC patient as well as transgenic mice showed decreased levels of p50 as well as TRUSS and accumulation of p105. Thus, enhanced expression of IL-10 gene in the presence of TRUSS and regulation of NF-κB1/p105 processing could be an important regulatory mechanism for inflammatory response and tumorgenic transformation.

Keywords:  E3 ubiquitin ligase; IL-10; TRUSS; Ubiquitination; p105; p50; p65

DOI:  https://doi.org/10.1016/j.cellsig.2020.109766
Autophagy. 2020 Sep 02.

The DNA methyltransferase DNMT3A contributes to autophagy long-term memory.

Patricia González-Rodríguez, Mathilde Cheray, Jens Füllgrabe, Maria Salli, Pinelopi Engskog-Vlachos, Lily Keane, Virginia Cunha, Agata Lupa, Wenbo Li, Qi Ma, Kristian Dreij, Michael G Rosenfeld, Bertrand Joseph.

  Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.

Keywords:  Autophagy; DNA methylation; MAP1LC3; epigenetics; transcription

DOI:  https://doi.org/10.1080/15548627.2020.1816664
Metabolism. 2020 Sep 01. pii: S0026-0495(20)30213-4. [Epub ahead of print] 154349

Hrd1-mediated ACLY Ubiquitination alleviate NAFLD in db/db mice.

Kai Li, Kaini Zhang, Hai Wang, Yangyang Wu, Nuoqi Chen, Jinfeng Chen, Chen Qiu, Pengpeng Cai, Min Li, Xiubin Liang, Dongming Su.

   BACKGROUND: The functions of Acly in regulating nonalcoholic fatty liver disease (NAFLD) have been identified; however, the dynamic control of Acly expression under the pathological state of metabolic disorders has not been fully elucidated. Previous studies reported an ubiquitin-proteasome-mediated degradation of Acly, but the mechanism is still largely unknown.
METHODS: Co-IP-based mass spectrum (MS/MS) assays were performed in HepG2 and Hepa1-6 hepatocytes and mouse liver tissue. The protein-protein interaction and ubiquitin modification of Hrd1 on Acly were confirmed by co-IP based immuno-blotting. Acetyl-CoA levels and lipogenesis rates were determined. The roles of Hrd1 on NAFLD and insulin resistance were tested by adenovirus-mediated overexpression in db/db mice or in separated primary hepatocytes.
RESULTS: Hrd1, a subunit of the endoplasmic reticulum-associated degradation (ERAD) complex, interacted with and ubiquitinated Acly, thereby reducing its protein level. Hrd1 suppressed the acetyl-CoA level and inhibited lipogenesis through an Acly-dependent pathway. The expression of hepatic Hrd1 was negatively associated with NAFLD, whereas overexpression of Hrd1 ameliorated hepatic steatosis and enhanced insulin sensitivity, both in db/db mice and in separated mouse primary hepatocytes.
CONCLUSIONS: Our results suggest that Acly, a master enzyme that regulates lipogenesis, is degraded by Hrd1 through ubiquitin modification. The activation of Hrd1 in hepatocytes might therefore represent a strategic approach for NAFLD therapy.

Keywords:  Acly stability; Hrd1; Insulin resistance; Liver steatosis; Ubiquitination

DOI:  https://doi.org/10.1016/j.metabol.2020.154349
Cell Chem Biol. 2020 Sep 01. pii: S2451-9456(20)30334-2. [Epub ahead of print]

Proteostasis Regulators Restore Function of Epilepsy-Associated GABAA Receptors.

Xiao-Jing Di, Ya-Juan Wang, Edmund Cotter, Meng Wang, Angela L Whittsette, Dong-Yun Han, Panjamaporn Sangwung, Renae Brown, Joseph W Lynch, Angelo Keramidas, Ting-Wei Mu.

  Proteostasis deficiency in mutated ion channels leads to a variety of ion channel diseases that are caused by excessive endoplasmic reticulum-associated degradation (ERAD) and inefficient membrane trafficking. We investigated proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors, the primary mediators of neuronal inhibition in the mammalian central nervous system. We screened a structurally diverse, Food and Drug Administration-approved drug library and identified dinoprost (DNP) and dihydroergocristine (DHEC) as highly efficacious enhancers of surface expression of four epilepsy-causing trafficking-deficient mutant receptors. Furthermore, DNP and DHEC restore whole-cell and synaptic currents by incorporating mutated subunits into functional receptors. Mechanistic studies revealed that both drugs reduce subunit degradation by attenuating the Grp94/Hrd1/Sel1L/VCP-mediated ERAD pathway and enhance the subunit folding by promoting subunit interactions with major GABAA receptors-interacting chaperones, BiP and calnexin. In summary, we report that DNP and DHEC remodel the endoplasmic reticulum proteostasis network to restore the functional surface expression of mutant GABAA receptors.

Keywords:  ERAD; GABA(A) receptors; assembly; chaperone; epilepsy; folding; misfolding; proteostasis; trafficking

DOI:  https://doi.org/10.1016/j.chembiol.2020.08.012
Fungal Biol. 2020 Sep;pii: S1878-6146(20)30095-7. [Epub ahead of print]124(9): 801-813

Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae.

Yuki Morita, Futa Kikumatsu, Yujiro Higuchi, Yoshinori Katakura, Kaoru Takegawa.

  Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.

Keywords:  AAA ATPase; Amylase; Aspergillus oryzae; Cdc48; ERAD

DOI:  https://doi.org/10.1016/j.funbio.2020.06.004
Theranostics. 2020 ;10(21): 9495-9511

DNA methylation downregulated ZDHHC1 suppresses tumor growth by altering cellular metabolism and inducing oxidative/ER stress-mediated apoptosis and pyroptosis.

Xin Le, Junhao Mu, Weiyan Peng, Jun Tang, Qin Xiang, Shaorong Tian, Yixiao Feng, Sanxiu He, Zhu Qiu, Guosheng Ren, Ailong Huang, Yong Lin, Qian Tao, Tingxiu Xiang.

  Cancer progression is an intricate biological process profiled by not only unscheduled proliferation, but also altered metabolism mechanisms. In this article, we introduced a novel tumor suppressor gene (TSG), Zinc Finger DHHC-Type Containing 1 (ZDHHC1, also known as ZNF377), frequently silenced due to epigenetic modification among various cancers, which exerts significant anti-tumor effects through metabolic regulation. Methods: Quantitative reversed-transcription PCR (qRT-PCR), reverse transcription PCR (RT-PCR) and Western blot were employed to demonstrate transcriptional and protein levels of targeted regulators. Methylation of ZDHHC1 promoter was detected by bisulfite genomic sequencing (BGS) and methylation specific PCR (MSP). Proteomics were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) and gas chromatography-mass spectrometry (GC-MS) were utilized for metabolomics analysis. Cellular functions were examined via corresponding approaches. Nude mice were used for xenograft tumor models. Indirect immunofluorescence staining was utilized to obtain precise location and expression of target proteins. Oxidative and ER stress indicators were detected using specific kits. Results: We found that ZDHHC1 expression was frequently silenced in multiple tumor cells and specimens due to methylation. Restoration of ZDHHC1 expression can curb cancer cell progression via stimulating apoptosis and cell cycle arrest, repressing metastasis, and reversing EMT transition and cell stemness. ZDHHC1's salient anti-tumor abilities were recognized in vivo as well. Metabolomic and proteomic analyses predicted inhibitory role of ZDHHC1 in glucose metabolism pathways in a CYGB-dependent manner, and in pentose phosphate pathway (PPP), which was validated by examining altered key factors. Moreover, we unraveled that ZDHHC1 dedicates to the increment of oxidative stress and endoplasmic reticulum (ER) stress to promote pyroptosis for anticancer purposes. Conclusion: Our study for the first time indicates ZDHHC1 is a potential tumor-suppressor frequently silenced due to promoter methylation, capable of negatively regulating metabolisms of tumor cells while stimulating oxidative stress and ER stress to expedite cell death through induction of pyroptosis and apoptosis, which can be exploited for development of new cancer prevention and therapies.

Keywords:  CYGB; ER stress; Oxidative stress; ZDHHC1; zinc finger protein

DOI:  https://doi.org/10.7150/thno.45631