bims-proteo 2020-07-05 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2020–07–05
thirty-six papers selected by
Eric Chevet, INSERM

Identification of a localized nonsense-mediated decay pathway at the endoplasmic reticulum.
The Unfolded Protein Response Regulates Hepatic Autophagy by sXBP1-mediated Activation of TFEB.
Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling.
TMEM16K is an interorganelle regulator of endosomal sorting.
ER-associated Protein Degradation at Atomic Resolution.
The ER chaperone calnexin controls mitochondrial positioning and respiration.
STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation.
Loss of ERAD bridging factor UBX2 modulates lipid metabolism and leads to ER stress-associated apoptosis during cadmium toxicity in Saccharomyces cerevisiae.
Protein modifications critical for myonectin/erythroferrone secretion and oligomer assembly.
A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.
Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells.
Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes.
Endocytic regulation of cellular ion homeostasis controls lysosome biogenesis.
The phosphoproteome response to dithiothreitol reveals unique versus shared features of Saccharomyces cerevisiae stress responses.
Unfolded protein response in cardiovascular disease.
Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs.
Vcp Overexpression and Leucine Supplementation Increase Protein Synthesis and Improve Fear Memory and Social Interaction of Nf1 Mutant Mice.
Systematic quantitative analysis of ribosome inventory during nutrient stress.
The proteostasis guardian HSF1 directs the transcription of its paralog and interactor HSF2 during proteasome dysfunction.
Wingless and Archipelago, a fly E3 ubiquitin ligase and a homolog of human tumor suppressor FBW7, show an antagonistic relationship in wing development.
Self-Association of Purified Reconstituted ER Luminal Spacer Climp63.
Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate.
Does proteostasis get lost in translation? Implications for protein aggregation across the lifespan.
Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport.
JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma.
LUNAPARK Is an E3 Ligase that Mediates Degradation of ROOT HAIR DEFECTIVE3 to Maintain a Tubular ER Network in Arabidopsis.
AMPK is activated during lysosomal damage via a galectin-ubiquitin signal transduction system.
Inhibition of Eukaryotic Translation Initiation Factor 5A (eIF5A) Hypusination Suppress p53 Translation and Alters the Association of eIF5A to the Ribosomes.
ULK1 and ULK2 are less redundant than previously thought: computational analysis uncovers distinct regulation and functions of these autophagy induction proteins.
Real-time monitoring of peroxiredoxin oligomerization dynamics in living cells.
Reciprocal regulation between GCN2 (eIF2AK4) and PERK (eIF2AK3) through the JNK-FOXO3 axis to modulate cancer drug resistance and clonal survival.
The Lotus japonicus Ubiquitin Ligase SIE3 Interacts With the Transcription Factor SIP1 and Forms a Homodimer.
Protein Sumoylation with SUMO1 Promoted by Pin1 in Glioma Stem Cells Augments Glioblastoma Malignancy.
HSP70 drives myoblast fusion during C2C12 myogenic differentiation.
Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress.
Molecular Basis of Ubiquitination Catalyzed by the Bacterial Transglutaminase MavC.

Genes Dev. 2020 Jul 02.

Identification of a localized nonsense-mediated decay pathway at the endoplasmic reticulum.

Dasa Longman, Kathryn A Jackson-Jones, Magdalena M Maslon, Laura C Murphy, Robert S Young, Jack J Stoddart, Nele Hug, Martin S Taylor, Dimitrios K Papadopoulos, Javier F Cáceres.

  Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.

Keywords:  ER stress; NBAS; RNA quality control; UPF1; UPR; nonsense-mediated decay (NMD)

DOI:  https://doi.org/10.1101/gad.338061.120
Autophagy. 2020 Jun 28.

The Unfolded Protein Response Regulates Hepatic Autophagy by sXBP1-mediated Activation of TFEB.

Zeyuan Zhang, Qingwen Qian, Mark Li, Fan Shao, Wen-Xing Ding, Vitor A Lira, Sophia X Chen, Sara C Sebag, Gökhan S Hotamisligil, Huojun Cao, Ling Yang.

  Defective macroautophagy/autophagy and a failure to initiate the adaptive unfolded protein response (UPR) in response to the endoplasmic reticulum (ER) stress contributes to obesity-associated metabolic dysfunction. However, whether and how unresolved ER stress leads to defects in the autophagy pathway and to the progression of obesity-associated hepatic pathologies remains unclear. Obesity suppresses the expression of hepatic spliced XBP1 (X-box binding protein 1; sXBP1), the key transcription factor that promotes the adaptive UPR. Our RNA-seq analysis revealed that sXBP1 regulates genes involved in lysosomal function in the liver under fasting conditions. Chromatin immunoprecipitation (ChIP) analyses of both primary hepatocytes and whole liver further showed that sXBP1 occupies the -743 to -523 site of the promoter of Tfeb (transcription factor EB), a master regulator of autophagy and lysosome biogenesis. Notably, this occupancy was significantly reduced in livers from patients with steatosis. In mice, hepatic deletion of Xbp1 (xbp1 LKO) suppressed the transcription of Tfeb as well as autophagy, whereas hepatic overexpression of sXbp1 enhanced Tfeb transcription and autophagy. Moreover, overexpression of Tfeb in the xbp1 LKO mouse liver ameliorated glucose intolerance and steatosis in mice with diet-induced obesity (DIO). Conversely, loss of TFEB function impaired the protective role of sXBP1 in hepatic steatosis in mice with DIO. These data indicate that sXBP1-Tfeb signaling has direct functional consequences in the context of obesity. Collectively, our data provide novel insight into how two organelle stress responses are integrated to protect against obesity-associated metabolic dysfunction.

Keywords:  autophagy; endoplasmic reticulum; liver; obesity; spliced X-box-binding protein 1; transcription factor EB

DOI:  https://doi.org/10.1080/15548627.2020.1788889
Elife. 2020 Jul 02. pii: e57306. [Epub ahead of print]9

Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling.

Emma J Fenech, Federica Lari, Philip D Charles, Roman Fischer, Marie Laétitia-Thézénas, Katrin Bagola, Adrienne W Paton, James C Paton, Mads Gyrd-Hansen, Benedikt M Kessler, John C Christianson.

  Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.

Keywords:  RNF26; STING; cell biology; endoplasmic reticulum; immunology; inflammation; innate immune response; none; ubiquitin ligase

DOI:  https://doi.org/10.7554/eLife.57306
Nat Commun. 2020 Jul 03. 11(1): 3298

TMEM16K is an interorganelle regulator of endosomal sorting.

Maja Petkovic, Juan Oses-Prieto, Alma Burlingame, Lily Yeh Jan, Yuh Nung Jan.

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime, where the endolysosomal pathway plays a prominent role by regulating protein and lipid sorting and degradation. Here we report that TMEM16K, an endoplasmic reticulum lipid scramblase causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with the small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting.

DOI: https://doi.org/10.1038/s41467-020-17016-8
Trends Biochem Sci. 2020 Jun 29. pii: S0968-0004(20)30152-3. [Epub ahead of print]

ER-associated Protein Degradation at Atomic Resolution.

Mohamed A Eldeeb, Richard P Fahlman, Marek Michalak.

  The endoplasmic reticulum-associated degradation (ERAD) pathway eliminates misfolded proteins. The Hrd1 complex represents the main gate mediating retrotranslocation of ER luminal misfolded (ERAD-L) substrates to the cytosol. A recent cryo-electron microscopy (cryo-EM) study by Wu et al. unveils the structural features of active Hrd1, providing mechanistic insights into the movement of proteins directed for degradation across ER membranes.

Keywords:  ERAD; HRD1 complex; cryo-EM; endoplasmic reticulum; proteasome; protein degradation; protein quality control; ubiquitin

DOI:  https://doi.org/10.1016/j.tibs.2020.06.005
Sci Signal. 2020 Jun 30. pii: eaax6660. [Epub ahead of print]13(638):

The ER chaperone calnexin controls mitochondrial positioning and respiration.

Tomás Gutiérrez, Hong Qi, Megan C Yap, Nasser Tahbaz, Leanne A Milburn, Eliana Lucchinetti, Phing-How Lou, Michael Zaugg, Paul G LaPointe, Pascal Mercier, Michael Overduin, Helmut Bischof, Sandra Burgstaller, Roland Malli, Klaus Ballanyi, Jianwei Shuai, Thomas Simmen.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.

DOI: https://doi.org/10.1126/scisignal.aax6660
Proc Natl Acad Sci U S A. 2020 Jun 29. pii: 201915386. [Epub ahead of print]

STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation.

Ga-Yeon Son, Krishna Prasad Subedi, Hwei Ling Ong, Lucile Noyer, Hassan Saadi, Changyu Zheng, Rajesh Bhardwaj, Stefan Feske, Indu Suresh Ambudkar.

  The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.

Keywords:  ER-PM junction; NFAT1; Orai1; STIM1; STIM2

DOI:  https://doi.org/10.1073/pnas.1915386117
Curr Genet. 2020 Jul 01.

Loss of ERAD bridging factor UBX2 modulates lipid metabolism and leads to ER stress-associated apoptosis during cadmium toxicity in Saccharomyces cerevisiae.

Selvaraj Rajakumar, Rajendran Vijayakumar, Albert Abhishek, Govindan Sadasivam Selvam, Vasanthi Nachiappan.

  The endoplasmic reticulum (ER) stress potentially activates the unfolded protein response (UPR) and ER-associated protein degradation (ERAD) as quality-control mechanisms. During ERAD process, the ERAD adaptor protein Ubx2 serves as a bridging factor and transports the misfolded proteins from the ER to the cytosol for subsequent ubiquitylation and proteasomal degradation. Cadmium (Cd) is a toxic metal that initiates ER stress and has an impact on lipid homeostasis and this study focuses on the synergistic impact of Cd exposure and ERAD (using ubx2∆ strain). With Cd exposure in ubx2∆ strain, we observed stunted growth and induction of ER stress. The ER stress was confirmed by measuring the expression of UPR marker (Kar2p), and mRNA expression of ER stress-responsive genes (HAC1, IRE1, ERO1, and PDI1), heat shock responsive genes (HSP104 and HSP60), ERAD pathway genes (DOA10, CDC48, HRD1, and YOS9), and proteasome regulators (UBI14, and RPN4). We also observed aberrant membrane morphology with DiOC6 staining, and interrupted mitochondria with mitotracker dye using microscopic analysis. The cell's inability to relieve stress through adaptive response results in apoptosis and was assessed using acridine orange (AO)-ethidium bromide (EtBr) staining. In ubx2∆ strain, there was reduction in triacylglycerol (TAG) and lipid droplets (LDs), and increase in the phospholipids. The mRNA expression of lipid metabolic genes (LRO1, DGA1, ARE1, ARE2, and OLE1) supported the lipid pattern observed. Collectively, our data suggest that in Saccharomyces cerevisiae, the Cd exposure on ubx2∆ strain induced cellular stress and has an impact on ERAD, UPR, and LD homeostasis.

Keywords:  ERAD; Lipid droplet and yeast; UPR

DOI:  https://doi.org/10.1007/s00294-020-01090-y
Biochemistry. 2020 Jun 30.

Protein modifications critical for myonectin/erythroferrone secretion and oligomer assembly.

Ashley N Stewart, Hannah C Little, David J Clark, Hui Zhang, G William Wong.

Myonectin/erythroferrone (also known as CTRP15) is a secreted hormone with metabolic function and a role in stress erythropoiesis. Despite its importance in physiologic processes, biochemical characterization of the protein is lacking. Here, we show that multiple protein modifications are critical for myonectin secretion and multimerization. Abolishing N-linked glycosylation by tunicamycin, glucosamine supplementation, or glutamine substitutions of all four potential Asn glycosylation sites blocked myonectin secretion. Mass spectrometry confirmed that Asn-229 and Asn-281 were glycosylated, and substituting both Asn sites with Gln prevented myonectin secretion. Although Asn-319 is not identified as glycosylated, Gln substitution caused protein misfolding and retention in the endoplasmic reticulum. Of the four conserved cysteines, Cys-273 and Cys-278 were required for proper protein folding; Ala substitution of either site inhibited protein secretion. In contrast, Ala substitutions of Cys-142, Cys-194, or both markedly enhanced protein secretion, suggesting endoplasmic reticulum retention that facilitates myonectin oligomer assembly. Secreted myonectin consists of trimers, hexamers, and high molecular weight (HMW) oligomers. Formation of higher-order structures via inter-molecular disulfide bonds depended on Cys-142 and Cys-194, whereas the C142A mutant formed almost exclusively trimers and the C194 mutant was impaired in HMW oligomer formation. Most Pro residues within the short collagen domain of myonectin were also hydroxylated, a modification that stabilized the collagen triple helix. Inhibiting Pro hydroxylation or deleting the collagen domain markedly reduced protein secretion. Together, our results reveal key determinants important for myonectin folding, secretion, and multimeric assembly and provide a basis for future structure/function studies.

DOI: https://doi.org/10.1021/acs.biochem.0c00461
J Clin Invest. 2020 Jun 29. pii: 130955. [Epub ahead of print]

A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.

Lakshya Bajaj, Jaiprakash Sharma, Alberto di Ronza, Pengcheng Zhang, Aiden Eblimit, Rituraj Pal, Dany Roman, John R Collette, Clarissa Booth, Kevin T Chang, Richard N Sifers, Sung Y Jung, Jill M Weimer, Rui Chen, Randy W Schekman, Marco Sardiello.

  Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.

Keywords:  Cell Biology; Genetic diseases; Lysosomes; Molecular pathology

DOI:  https://doi.org/10.1172/JCI130955
Proc Natl Acad Sci U S A. 2020 Jun 29. pii: 202004606. [Epub ahead of print]

Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells.

Hiroshi Kadokura, Yui Dazai, Yo Fukuda, Naoya Hirai, Orie Nakamura, Kenji Inaba.

  Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.

Keywords:  LDL receptor; cotranslational folding; disulfide bonds; multidomain protein; nonvectorial folding

DOI:  https://doi.org/10.1073/pnas.2004606117
Nat Commun. 2020 Jul 03. 11(1): 3306

Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes.

Keisuke Mochida, Akinori Yamasaki, Kazuaki Matoba, Hiromi Kirisako, Nobuo N Noda, Hitoshi Nakatogawa.

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.

DOI: https://doi.org/10.1038/s41467-020-17163-y
Nat Cell Biol. 2020 Jun 29.

Endocytic regulation of cellular ion homeostasis controls lysosome biogenesis.

Tania López-Hernández, Dmytro Puchkov, Eberhard Krause, Tanja Maritzen, Volker Haucke.

Lysosomes serve as cellular degradation and signalling centres that coordinate metabolism in response to intracellular cues and extracellular signals. Lysosomal capacity is adapted to cellular needs by transcription factors, such as TFEB and TFE3, which activate the expression of lysosomal and autophagy genes. Nuclear translocation and activation of TFEB are induced by a variety of conditions such as starvation, lysosome stress and lysosomal storage disorders. How these various cues are integrated remains incompletely understood. Here, we describe a pathway initiated at the plasma membrane that controls lysosome biogenesis via the endocytic regulation of intracellular ion homeostasis. This pathway is based on the exo-endocytosis of NHE7, a Na+/H+ exchanger mutated in X-linked intellectual disability, and serves to control intracellular ion homeostasis and thereby Ca2+/calcineurin-mediated activation of TFEB and downstream lysosome biogenesis in response to osmotic stress to promote the turnover of toxic proteins and cell survival.

DOI: https://doi.org/10.1038/s41556-020-0535-7
J Proteome Res. 2020 Jun 29.

The phosphoproteome response to dithiothreitol reveals unique versus shared features of Saccharomyces cerevisiae stress responses.

Matthew E MacGilvray, Evgenia Shishkova, Michael Place, Ellen R Wagner, Joshua J Coon, Audrey P Gasch.

To cope with sudden changes in the external environment, the budding yeast Saccharomyces cerevisiae orchestrates a multi-faceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factor. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent DTT and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking IRE1 or MAPK kinases MKK1 and MKK2, before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of Protein Kinase A (PKA) targets. These results have implications for the breadth of signaling programs that can give rise to common stress response signatures.

DOI: https://doi.org/10.1021/acs.jproteome.0c00253
Cell Signal. 2020 Jun 25. pii: S0898-6568(20)30176-5. [Epub ahead of print]73 109699

Unfolded protein response in cardiovascular disease.

Khadeja-Tul Kubra, Mohammad S Akhter, Mohammad A Uddin, Nektarios Barabutis.

  The unfolded protein response (UPR) is a highly conserved molecular machinery, which protects the cells against a diverse variety of stimuli. Activation of this element has been associated with both human health and disease. The purpose of the current manuscript is to provide the most updated information on the involvement of UPR towards the improvement; or deterioration of cardiovascular functions. Since UPR is consisted of three distinct elements, namely the activating transcription factor 6, the protein kinase RNA-like endoplasmic reticulum kinase; and the inositol-requiring enzyme-1α, a highly orchestrated manipulation of those molecular branches may provide new therapeutic possibilities against the severe outcomes of cardiovascular disease.

Keywords:  Arrhythmia; Cardiac hypertrophy; ER stress; Myocardial infarction

DOI:  https://doi.org/10.1016/j.cellsig.2020.109699
Sci Rep. 2020 Jul 01. 10(1): 10733

Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs.

Anna Kaziales, Katalin Barkovits, Katrin Marcus, Klaus Richter.

The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR·Hsp90·Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems.

DOI: https://doi.org/10.1038/s41598-020-67645-8
Cell Rep. 2020 Jun 30. pii: S2211-1247(20)30816-0. [Epub ahead of print]31(13): 107835

Vcp Overexpression and Leucine Supplementation Increase Protein Synthesis and Improve Fear Memory and Social Interaction of Nf1 Mutant Mice.

Yu-Tzu Shih, Tzyy-Nan Huang, Hsiao-Tang Hu, Tzu-Li Yen, Yi-Ping Hsueh.

  Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/- mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/- neurons. Contextual memory and social behavior of Nf1+/- mice are also restored by leucine supplementation. Our study suggests that the "ER-protein synthesis" pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.

Keywords:  autism spectrum disorders; branched-chain amino acids; dendritic spine formation; endoplasmic reticulum; leucine; neurodevelopmental disorders; neurofibromatosis type 1; neurofibromin; valosin-containing protein/P97

DOI:  https://doi.org/10.1016/j.celrep.2020.107835
Nature. 2020 Jul 01.

Systematic quantitative analysis of ribosome inventory during nutrient stress.

Heeseon An, Alban Ordureau, Maria Körner, Joao A Paulo, J Wade Harper.

Mammalian cells reorganize their proteomes in response to nutrient stress through translational suppression and degradative mechanisms using the proteasome and autophagy systems1,2. Ribosomes are central targets of this response, as they are responsible for translation and subject to lysosomal turnover during nutrient stress3-5. The abundance of ribosomal (r)-proteins (around 6% of the proteome; 107 copies per cell)6,7 and their high arginine and lysine content has led to the hypothesis that they are selectively used as a source of basic amino acids during nutrient stress through autophagy4,7. However, the relative contributions of translational and degradative mechanisms to the control of r-protein abundance during acute stress responses is poorly understood, as is the extent to which r-proteins are used to generate amino acids when specific building blocks are limited7. Here, we integrate quantitative global translatome and degradome proteomics8 with genetically encoded Ribo-Keima5 and Ribo-Halo reporters to interrogate r-protein homeostasis with and without active autophagy. In conditions of acute nutrient stress, cells strongly suppress the translation of r-proteins, but, notably, r-protein degradation occurs largely through non-autophagic pathways. Simultaneously, the decrease in r-protein abundance is compensated for by a reduced dilution of pre-existing ribosomes and a reduction in cell volume, thereby maintaining the density of ribosomes within single cells. Withdrawal of basic or hydrophobic amino acids induces translational repression without differential induction of ribophagy, indicating that ribophagy is not used to selectively produce basic amino acids during acute nutrient stress. We present a quantitative framework that describes the contributions of biosynthetic and degradative mechanisms to r-protein abundance and proteome remodelling in conditions of nutrient stress.

DOI: https://doi.org/10.1038/s41586-020-2446-y
Cell Mol Life Sci. 2020 Jun 30.

The proteostasis guardian HSF1 directs the transcription of its paralog and interactor HSF2 during proteasome dysfunction.

Silvia Santopolo, Anna Riccio, Antonio Rossi, M Gabriella Santoro.

  Protein homeostasis is essential for life in eukaryotes. Organisms respond to proteotoxic stress by activating heat shock transcription factors (HSFs), which play important roles in cytoprotection, longevity and development. Of six human HSFs, HSF1 acts as a proteostasis guardian regulating stress-induced transcriptional responses, whereas HSF2 has a critical role in development, in particular of brain and reproductive organs. Unlike HSF1, that is a stable protein constitutively expressed, HSF2 is a labile protein and its expression varies in different tissues; however, the mechanisms regulating HSF2 expression remain poorly understood. Herein we demonstrate that the proteasome inhibitor anticancer drug bortezomib (Velcade), at clinically relevant concentrations, triggers de novo HSF2 mRNA transcription in different types of cancers via HSF1 activation. Similar results were obtained with next-generation proteasome inhibitors ixazomib and carfilzomib, indicating that induction of HSF2 expression is a general response to proteasome dysfunction. HSF2-promoter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies unexpectedly revealed that HSF1 is recruited to a heat shock element located at 1.397 bp upstream from the transcription start site in the HSF2-promoter. More importantly, we found that HSF1 is critical for HSF2 gene transcription during proteasome dysfunction, representing an interesting example of transcription factor involved in controlling the expression of members of the same family. Moreover, bortezomib-induced HSF2 was found to localize in the nucleus, interact with HSF1, and participate in bortezomib-mediated control of cancer cell migration. The results shed light on HSF2-expression regulation, revealing a novel level of HSF1/HSF2 interplay that may lead to advances in pharmacological modulation of these fundamental transcription factors.

Keywords:  Anticancer; Bortezomib; Cell migration; HSF1; HSF2; Proteasome inhibition; Transcriptional regulation

DOI:  https://doi.org/10.1007/s00018-020-03568-x
BMC Dev Biol. 2020 Jun 29. 20(1): 14

Wingless and Archipelago, a fly E3 ubiquitin ligase and a homolog of human tumor suppressor FBW7, show an antagonistic relationship in wing development.

Sujin Nam, Kyung-Ok Cho.

   BACKGROUND: Archipelago (Ago) is a Drosophila homolog of mammalian F-box and WD repeat domain-containing 7 (FBW7, also known as FBXW7). In previous studies, FBW7 has been addressed as a tumor suppressor mediating ubiquitin-dependent proteolysis of several oncogenic proteins. Ubiquitination is a type of protein modification that directs protein for degradation as well as sorting. The level of beta-catenin (β-cat), an intracellular signal transducer in Wnt signaling pathway, is reduced upon overexpression of FBW7 in human cancer cell lines. Loss of function mutations in FBW7 and overactive Wnt signaling have been reported to be responsible for human cancers.
RESULTS: We found that Ago is important for the formation of shafts in chemosensory bristles at wing margin. This loss of shaft phenotype by knockdown of ago was rescued by knockdown of wingless (wg) whereas wing notching phenotype by knockdown of wg was rescued by knockdown of ago, establishing an antagonistic relationship between ago and wg. In line with this finding, knockdown of ago increased the level of Armadillo (Arm), a homolog of β-cat, in Drosophila tissue. Furthermore, knockdown of ago increased the level of Distal-less (Dll) and extracellular Wg in wing discs. In S2 cells, the amount of secreted Wg was increased by knockdown of Ago but decreased by Ago overexpression. Therefore, Ago plays a previously unidentified role in the inhibition of Wg secretion. Ago-overexpressing clones in wing discs exhibited accumulation of Wg in endoplasmic reticulum (ER), suggesting that Ago prevents Wg protein from moving to Golgi from ER.
CONCLUSIONS: We concluded that Ago plays dual roles in inhibiting Wg signaling. First, Ago decreases the level of Arm, by which Wg signaling is downregulated in Wg-responding cells. Second, Ago decreases the level of extracellular Wg by inhibiting movement of Wg from ER to Golgi in Wg-producing cells.

Keywords:  Ago; Archipelago; Chemosensory bristles; E3 ubiquitin ligase; FBW7; Shaft; Wg secretion; Wg signaling

DOI:  https://doi.org/10.1186/s12861-020-00217-1
Front Cell Dev Biol. 2020 ;8 500

Self-Association of Purified Reconstituted ER Luminal Spacer Climp63.

Jinghua Zhao, Junjie Hu.

  Membranes of the endoplasmic reticulum (ER) are shaped into cisternal sheets and cylindrical tubules. How ER sheets are generated and maintained is not clear. ER membrane protein Climp63 is enriched in sheets and routinely used as a marker of this structure. The luminal domain (LD) of Climp63 is predicted to be highly helical, and it may form bridges between parallel membranes, regulating the abundance and width of ER sheets. Here, we purified the LD and full-length (FL) Climp63 to analyze their homotypic interactions. The N-terminal tagged LD formed low-order oligomers in solution, but was extremely aggregation-prone when the GST tag was removed. Purified FL Climp63 formed detectable but moderate interactions with both the FL protein and the LD. When Climp63 was reconstituted into proteoliposomes with its LD facing out, the homotypic interactions were retained and could be competed by soluble LD, though vesicle clustering was not observed. These results demonstrate a direct self-association of Climp63, supporting its role as an ER luminal spacer.

Keywords:  Climp63; endoplasmic reticulum; homotypic interactions; membrane tethering; reconstitution; sheet biogenesis

DOI:  https://doi.org/10.3389/fcell.2020.00500
Cell. 2020 Jun 23. pii: S0092-8674(20)30693-0. [Epub ahead of print]

Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate.

Colin Chih-Chien Wu, Amy Peterson, Boris Zinshteyn, Sergi Regot, Rachel Green.

  Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.

Keywords:  SAPK; UV radiation; ZAK; amino acid starvation; integrated stress response; ribosome collisions

DOI:  https://doi.org/10.1016/j.cell.2020.06.006
Ageing Res Rev. 2020 Jun 27. pii: S1568-1637(20)30254-3. [Epub ahead of print] 101119

Does proteostasis get lost in translation? Implications for protein aggregation across the lifespan.

Stephany Francisco, Margarida Ferreira, Gabriela Moura, Ana Raquel Soares, Manuel A S Santos.

  Protein aggregation is a phenomenon of major relevance in neurodegenerative and neuromuscular disorders, cataracts, diabetes and many other diseases. Research has unveiled that proteins also aggregate in multiple tissues during healthy aging yet, the biological and biomedical relevance of this apparently asymptomatic phenomenon remains to be understood. It is known that proteome homeostasis (proteostasis) is maintained by a balanced protein synthesis rate, high protein synthesis accuracy, efficient protein folding and continual tagging of damaged proteins for degradation, suggesting that protein aggregation during healthy aging may be associated with alterations in both protein synthesis and the proteostasis network (PN) pathways. In particular, dysregulation of protein synthesis and alterations in translation fidelity are hypothesized to lead to the production of misfolded proteins which could explain the occurrence of age-related protein aggregation. Nevertheless, some data on this topic is controversial and the biological mechanisms that lead to widespread protein aggregation remain to be elucidated. We review the recent literature about the age-related decline of proteostasis, highlighting the need to build an integrated view of protein synthesis rate, fidelity and quality control pathways in order to better understand the proteome alterations that occur during aging and in age-related diseases.

Keywords:  Aging; Protein aggregation; Protein synthesis; Proteostasis network; Translation fidelity; Translation rate

DOI:  https://doi.org/10.1016/j.arr.2020.101119
J Cell Sci. 2020 Jul 02. pii: jcs.239814. [Epub ahead of print]

Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport.

L M Westrate, M J Hoyer, M J Nash, G K Voeltz.

  Secretory cargo is recognized, concentrated and trafficked from ER exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo loaded, COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII coated vesicle. Here, we utilized a recently developed live cell, cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We find that components of the COPII coat remain associated with the ERES, while cargo is extruded into COPII uncoated, non-ER associated, Rab1-dependent carriers. Our data suggest that in animal cells COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.

Keywords:  COPII; ERES; MANII; RUSH; Rab1; TNF-alpha

DOI:  https://doi.org/10.1242/jcs.239814
Proc Natl Acad Sci U S A. 2020 Jun 29. pii: 202002672. [Epub ahead of print]

JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma.

Elisa Manieri, Cintia Folgueira, María Elena Rodríguez, Luis Leiva-Vega, Laura Esteban-Lafuente, Chaobo Chen, Francisco Javier Cubero, Tamera Barrett, Julie Cavanagh-Kyros, Davide Seruggia, Alejandro Rosell, Fátima Sanchez-Cabo, Manuel Jose Gómez, Maria J Monte, Jose J G Marin, Roger J Davis, Alfonso Mora, Guadalupe Sabio.

  Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.

Keywords:  JNK; PPARa; bile acid; cholangiocarcinoma

DOI:  https://doi.org/10.1073/pnas.2002672117
Plant Cell. 2020 Jul 02. pii: tpc.00937.2018. [Epub ahead of print]

LUNAPARK Is an E3 Ligase that Mediates Degradation of ROOT HAIR DEFECTIVE3 to Maintain a Tubular ER Network in Arabidopsis.

Jiaqi Sun, Nooshin Movahed, Huanquan Zheng.

RHD3 (ROOT HAIR DEFECTIVE3) is an atlastin GTPase involved in homotypic fusion of ER tubules in the formation of the interconnected ER network. Because excessive fusion of ER tubules will lead to the formation of sheet-like ER, the action of atlastin GTPases must be tightly regulated. We show here that RHD3 physically interacts with two Arabidopsis LUNAPARK proteins (LNP1 and LNP2) at 3-way junctions of the ER, the sites where different ER tubules fuse. Recruited by RHD3 to newly formed 3-way junctions, LNPs act negatively with RHD3 to stabilize the nascent 3-way junctions of the ER. Without this LNP-mediated stabilization, in Arabidopsis lnp1-1 lnp2-1 mutant cells, the ER becomes a dense tubular network. Interestingly, in lnp1-1 lnp2-1 mutant cells, the RHD3 level is higher than that in wild type plants. RHD3 is degraded more slowly in the absence of LNPs, as well as in the presence of MG132 and Concanamycin A. However, in the presence of LNPs, the degradation of RHD3 is promoted. We have provided an in vitro evidence that Arabidopsis LNPs have E3 ubiquitin ligase activity and that LNP1 can directly ubiquitinates RHD3. Our data show that after ER fusion is completed, RHD3 is degraded by LNPs so that nascent 3-way junctions can be stabilized and a tubular ER network can be maintained.

DOI: https://doi.org/10.1105/tpc.18.00937
Autophagy. 2020 Jun 28.

AMPK is activated during lysosomal damage via a galectin-ubiquitin signal transduction system.

Jingyue Jia, Bhawana Bissa, Lukas Brecht, Lee Allers, Seong Won Choi, Yuexi Gu, Mark Zbinden, Mark R Burge, Graham Timmins, Kenneth Hallows, Christian Behrends, Vojo Deretic.

  Lysosomal damage activates AMPK, a regulator of macroautophagy/autophagy and metabolism, and elicits a strong ubiquitination response. Here we show that the cytosolic lectin LGALS9 detects lysosomal membrane breach by binding to lumenal glycoepitopes, and directs both the ubiquitination response and AMPK activation. Proteomic analyses have revealed increased LGALS9 association with lysosomes, and concomitant changes in LGALS9 interactions with its newly identified partners that control ubiquitination-deubiquitination processes. An LGALS9-inetractor, deubiquitinase USP9X, dissociates from damaged lysosomes upon recognition of lumenal glycans by LGALS9. USP9X's departure from lysosomes promotes K63 ubiquitination and stimulation of MAP3K7/TAK1, an upstream kinase and activator of AMPK hitherto orphaned for a precise physiological function. Ubiquitin-activated MAP3K7/TAK1 controls AMPK specifically during lysosomal injury, caused by a spectrum of membrane-damaging or -permeabilizing agents, including silica crystals, the intracellular pathogen Mycobacterium tuberculosis, TNFSF10/TRAIL signaling, and the anti-diabetes drugs metformin. The LGALS9-ubiquitin system activating AMPK represents a novel signal transduction system contributing to various physiological outputs that are under the control of AMPK, including autophagy, MTOR, lysosomal maintenance and biogenesis, immunity, defense against microbes, and metabolic reprograming.

Keywords:   Mycobacterium tuberculosis ; AMPK; TAK1; TRAIL; USP9X; autophagy; diabetes; lysosome; metabolism; metformin

DOI:  https://doi.org/10.1080/15548627.2020.1788890
Int J Mol Sci. 2020 Jun 27. pii: E4583. [Epub ahead of print]21(13):

Inhibition of Eukaryotic Translation Initiation Factor 5A (eIF5A) Hypusination Suppress p53 Translation and Alters the Association of eIF5A to the Ribosomes.

Marianna Martella, Caterina Catalanotto, Claudio Talora, Anna La Teana, Paola Londei, Dario Benelli.

  The eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for the viability of the cells whose proposed function is to prevent the stalling of the ribosomes during translation elongation. eIF5A activity requires a unique and functionally essential post-translational modification, the change of a lysine to hypusine. eIF5A is recognized as a promoter of cell proliferation, but it has also been suggested to induce apoptosis. To date, the precise molecular mechanism through which eIF5A affects these processes remains elusive. In the present study, we explored whether eIF5A is involved in controlling the stress-induced expression of the key cellular regulator p53. Our results show that treatment of HCT-116 colon cancer cells with the deoxyhypusine (DHS) inhibitor N1-guanyl-1,7-diamineheptane (GC7) caused both inhibition of eIF5A hypusination and a significant reduction of p53 expression in UV-treated cells, and that eIF5A controls p53 expression at the level of protein synthesis. Furthermore, we show that treatment with GC7 followed by UV-induced stress counteracts the pro-apoptotic process triggered by p53 up-regulation. More in general, the importance of eIF5A in the cellular stress response is illustrated by the finding that exposure to UV light promotes the binding of eIF5A to the ribosomes, whereas UV treatment complemented by the presence of GC7 inhibits such binding, allowing a decrease of de novo synthesis of p53 protein.

Keywords:  GC7; colon cancer cell lines; eIF5A; p53

DOI:  https://doi.org/10.3390/ijms21134583
Sci Rep. 2020 Jul 02. 10(1): 10940

ULK1 and ULK2 are less redundant than previously thought: computational analysis uncovers distinct regulation and functions of these autophagy induction proteins.

Amanda Demeter, Mari Carmen Romero-Mulero, Luca Csabai, Márton Ölbei, Padhmanand Sudhakar, Wilfried Haerty, Tamás Korcsmáros.

Macroautophagy, the degradation of cytoplasmic content by lysosomal fusion, is an evolutionary conserved process promoting homeostasis and intracellular defence. Macroautophagy is initiated primarily by a complex containing ULK1 or ULK2 (two paralogs of the yeast Atg1 protein). To understand the differences between ULK1 and ULK2, we compared the human ULK1 and ULK2 proteins and their regulation. Despite the similarity in their enzymatic domain, we found that ULK1 and ULK2 have major differences in their autophagy-related interactors and their post-translational and transcriptional regulators. We identified 18 ULK1-specific and 7 ULK2-specific protein motifs serving as different interaction interfaces. We found that interactors of ULK1 and ULK2 all have different tissue-specific expressions partially contributing to diverse and ULK-specific interaction networks in various tissues. We identified three ULK1-specific and one ULK2-specific transcription factor binding sites, and eight sites shared by the regulatory region of both genes. Importantly, we found that both their post-translational and transcriptional regulators are involved in distinct biological processes-suggesting separate functions for ULK1 and ULK2. Unravelling differences between ULK1 and ULK2 could lead to a better understanding of how ULK-type specific dysregulation affects autophagy and other cellular processes that have been implicated in diseases such as inflammatory bowel disease and cancer.

DOI: https://doi.org/10.1038/s41598-020-67780-2
Proc Natl Acad Sci U S A. 2020 Jun 29. pii: 201915275. [Epub ahead of print]

Real-time monitoring of peroxiredoxin oligomerization dynamics in living cells.

Daniel Pastor-Flores, Deepti Talwar, Brandán Pedre, Tobias P Dick.

  Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.

Keywords:  dimer-decamer equilibrium; fluorescence polarization; homo-FRET; peroxiredoxins; protein oligomerization

DOI:  https://doi.org/10.1073/pnas.1915275117
Mol Cell Endocrinol. 2020 Jun 29. pii: S0303-7207(20)30232-X. [Epub ahead of print] 110932

Reciprocal regulation between GCN2 (eIF2AK4) and PERK (eIF2AK3) through the JNK-FOXO3 axis to modulate cancer drug resistance and clonal survival.

Glowi Alasiri, Yannasittha Jiramongkol, Sasanan Trakansuebkul, Hui-Ling Ke, Zimam Mahmud, Kitti Intuyod, Eric W-F Lam.

  Pharmaceutical inhibitors of the endoplasmic reticulum (ER)-stress modulator PERK (eIF2AK3) have demonstrated anticancer activities in combination therapies, but their effectiveness as a single agent is limited, suggesting the existence of possible compensatory cellular responses. To explore the potential mechanisms involved, we performed time-course drug treatment experiments on the parental MCF-7 and drug resistant MCF-7EpiR and MCF-7TaxR breast cancer cells and identified GCN2 (eIF2AK4) as a molecule that can potentially cooperate with PERK to regulate FOXO3 via JNK and AKT to modulate drug response. Consistently, GCN2 knockdown severely impaired the clonal survival of parental and resistant MCF-7 cells and sensitised them to epirubicin and paclitaxel treatment. Western blot, RT-qPCR and ChIP analyses also confirmed that GCN2 inactivation causes an induction of JNK and thereby FOXO3 activity, culminating in an increase in PERK activity and expression at the transcription level. Conversely, PERK-inactivation using GSK2606414 also induces an induction in GCN2 expression and activity also associated with JNK. In agreement, we also showed that the perk-/- MEFs, expressing elevated levels of P-JNK, JNK, GCN2 and reduced levels of P-AKT and P-FOXO3, have lower clonogenicity and are more sensitive to epirubicin compared to wild-type MEFs. Similarly, gcn2-/- MEFs expressing augmented levels of P-JNK, JNK, P-PERK, PERK and lower levels of P-AKT and P-FOXO3 also had lower clonogenicity and were more sensitive to epirubicin and PERK-inhibition. In addition, JNK1/2 deletion in MEFs resulted in reduced levels of GCN2, FOXO3, PERK, P-PERK expression as well as FOXO3 activity and enhanced clonal survival and resistance to PERK-inhibition. Together these results demonstrate that GCN2 cooperates with PERK through the JNK-FOXO3 axis in a reciprocal negative feedback loop to mediate cancer chemotherapeutic drug response and clonal survival, advocating the potential of targeting GCN2 as a therapeutic strategy for treating cancer and for overcoming drug resistance.

Keywords:  AKT; Breast cancer; Chemotherapy; FOXO3; Forkhead transcription factor; JNK

DOI:  https://doi.org/10.1016/j.mce.2020.110932
Front Plant Sci. 2020 ;11 795

The Lotus japonicus Ubiquitin Ligase SIE3 Interacts With the Transcription Factor SIP1 and Forms a Homodimer.

Yong Feng, Ping Wu, Weiwei Fu, Liwei Peng, Hui Zhu, Yangrong Cao, Xinan Zhou, Zonglie Hong, Zhongming Zhang, Songli Yuan.

  The symbiosis receptor kinase SymRK plays an essential role in symbiotic signal transduction and nodule organogenesis. Several proteins bind to SymRK, but how the symbiosis signals are transduced from SymRK to downstream components remains elusive. We previously demonstrated that both SymRK interacting protein 1 (SIP1, an ARID-type DNA-binding protein) and SymRK interacting E3 ligase [SIE3, a RING (Really Interesting New Gene)-containing E3 ligase] interact with SymRK to regulate downstream cellular responses in Lotus japonicus during the legume-rhizobia symbiosis. Here, we show that SIE3 interacts with SIP1 in both yeast cells and Nicotiana benthamiana. SIE3 associated with itself and formed a homodimer. The cysteine 266 residue was found to be essential for SIE3 dimerization and for promoting nodulation in transgenic hairy roots of L. japonicus. Our findings provide a foundation for further investigating the regulatory mechanisms of the SymRK-mediated signaling pathway, as well as the biological function of E3 ligase dimerization in nodule organogenesis.

Keywords:  SIE3; SIP1; protein dimerization; rhizobium-legume symbiosis; symRK; symbiosis signaling

DOI:  https://doi.org/10.3389/fpls.2020.00795
Neuro Oncol. 2020 Jun 27. pii: noaa150. [Epub ahead of print]

Protein Sumoylation with SUMO1 Promoted by Pin1 in Glioma Stem Cells Augments Glioblastoma Malignancy.

Aili Zhang, Weiwei Tao, Kui Zhai, Xiaoguang Fang, Zhi Huang, Jennifer S Yu, Andrew E Solan, Jeremy N Rich, Wenchao Zhou, Shideng Bao.

   BACKGROUND: The tumorigenic potential of glioma stem cells (GSCs) is associated with multiple reversible molecular alternations, but the role of post-translational protein sumoylation in GSCs has not been elucidated. The development of GSC-targeting drugs relies on the discovery of GSC-preferential molecular modifications and the relevant signaling pathways. In this work, we investigated the protein sumoylation status, the major sumoylated substrate and the key regulatory enzyme in GSCs to explore the therapeutic potential of disrupting protein sumoylation for glioblastoma (GBM) treatment.
METHODS: Patient-derived GSCs, primary GBM sections, and intracranial GBM xenografts were used to determine protein sumoylation and the related molecular mechanisms by immunoblot, quantitative PCR, immunoprecipitation, immunofluorescence and immunohistochemistry. Orthotopic GBM xenograft models were applied to investigate the inhibition of tumor growth by disrupting protein sumoylation with shRNAs or molecular inhibitors.
RESULTS: We show that high levels of SUMO1- but not SUMO2/3-modified sumoylation are preferentially present in GSCs. The promyelocytic leukaemia protein (PML) is a major SUMO1-sumoylated substrate in GSCs, whose sumoylation facilitates its interaction with c-Myc to stabilize c-Myc proteins. The prolyl-isomerase Pin1 is preferentially expressed in GSCs and functions as the key enzyme to promote SUMO1-sumoylation. Disruption of SUMO1-sumoylation by Pin1 silencing with shRNAs or inhibition with its inhibitor Juglone markedly abrogated GSC maintenance and mitigated GSC-driven tumor growth.
CONCLUSIONS: Our findings indicate that high SUMO1-modified protein sumoylation as a feature of GSCs is critical for GSC maintenance, suggesting that targeting SUMO1-sumoylation may effectively improve GBM treatment.

Keywords:  Glioma stem cells; PML; Pin1; Post-translational modification; Protein sumoylation

DOI:  https://doi.org/10.1093/neuonc/noaa150
Biol Open. 2020 Jun 30. pii: bio.053918. [Epub ahead of print]

HSP70 drives myoblast fusion during C2C12 myogenic differentiation.

Savant S Thakur, Kristy Swiderski, Victoria L Chhen, Janine L James, Nicki J Cranna, A M Taufiqual Islam, James G Ryall, Gordon S Lynch.

  In response to injury, skeletal muscle stem cells (MuSCs) undergo myogenesis where they become activated, proliferate rapidly, differentiate and undergo fusion to form multinucleated myotubes. Dramatic changes in cell size, shape, metabolism and motility occur during myogenesis which cause cellular stress and alter proteostasis. The molecular chaperone heat shock protein 70 (HSP70) maintains proteostasis by regulating protein biosynthesis and folding, facilitating transport of polypeptides across intracellular membranes and preventing stress-induced protein unfolding/aggregation. Although HSP70 overexpression can exert beneficial effects in skeletal muscle diseases and enhance skeletal muscle repair after injury, its effect on myogenesis has not been investigated. Plasmid-mediated overexpression of HSP70 did not affect the rate of C2C12 proliferation or differentiation, but the median number of myonuclei per myotube and median myotube width in differentiated C2C12 myotubes were increased with HSP70 overexpression. These findings reveal that increased HSP70 expression can promote myoblast fusion, identifying a mechanism for its therapeutic potential to enhance muscle repair after injury.

Keywords:  C2C12; Fusion; Heat shock protein 70; Myogenesis; Skeletal muscle

DOI:  https://doi.org/10.1242/bio.053918
Sci Rep. 2020 Jul 03. 10(1): 10986

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress.

Sarah A White, Lisa S Zhang, Daniel J Pasula, Yu Hsuan Carol Yang, Dan S Luciani.

ER stress and apoptosis contribute to the loss of pancreatic β-cells under pro-diabetic conditions of glucolipotoxicity. Although activation of canonical intrinsic apoptosis is known to require pro-apoptotic Bcl-2 family proteins Bax and Bak, their individual and combined involvement in glucolipotoxic β-cell death are not known. It has also remained an open question if Bax and Bak in β-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined β-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic β-cell death in vitro occurs by both non-apoptotic and apoptotic mechanisms, and the apoptosis could be triggered by either Bax or Bak alone. In contrast, they had non-redundant roles in mediating staurosporine-induced apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated β-cell Ca2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that combined deletion of Bax and Bak amplified the unfolded protein response in islets during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings shed new light on roles of the core apoptosis machinery in β-cell survival and stress signals of importance for the pathobiology of diabetes.

DOI: https://doi.org/10.1038/s41598-020-67755-3
Adv Sci (Weinh). 2020 Jun;7(12): 2000871

Molecular Basis of Ubiquitination Catalyzed by the Bacterial Transglutaminase MavC.

Hongxin Guan, Jiaqi Fu, Ting Yu, Zhao-Xi Wang, Ninghai Gan, Yini Huang, Vanja Perčulija, Yu Li, Zhao-Qing Luo, Songying Ouyang.

  The Legionella pneumophila effector MavC is a transglutaminase that carries out atypical ubiquitination of the host ubiquitin (Ub)-conjugation enzyme UBE2N by catalyzing the formation of an isopeptide bond between Gln40Ub and Lys92UBE2N, which leads to inhibition of signaling in the NF-κB pathway. In the absence of UBE2N, MavC deamidates Ub at Gln40 or catalyzes self-ubiquitination. However, the mechanisms underlying these enzymatic activities of MavC are poorly understood at the molecular level. This study reports the structure of the MavC-UBE2N-Ub ternary complex representing a snapshot of MavC-catalyzed crosslinking of UBE2N and Ub, which reveals the way by which UBE2N-Ub binds to the Insertion and Tail domains of MavC. Based on the structural and experimental data, the catalytic mechanism for the deamidase and transglutaminase activities of MavC is proposed. Finally, by comparing the structures of MavC and MvcA, the homologous protein that reverses MavC-induced UBE2N ubiquitination, several essential regions and two key residues (Trp255MavC and Phe268MvcA) responsible for their respective enzymatic activities are identified. The results provide insights into the mechanisms for substrate recognition and ubiquitination mediated by MavC as well as explanations for the opposite activity of MavC and MvcA in terms of regulation of UBE2N ubiquitination.

Keywords:  Legionella pneumophila; NF‐κB; deamidase; effectors; transglutamination

DOI:  https://doi.org/10.1002/advs.202000871