bims-proteo 2021-08-08 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2021–08–08
twenty-two papers selected by
Eric Chevet, INSERM

Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy, and Collagen quality control.
Dual topology of co-chaperones at the membrane of the endoplasmic reticulum.
The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells.
STING1 is essential for an RNA-virus triggered autophagy.
Are cachexia-associated tumors transmitTERS of ER stress?
A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities.
The E3 ubiquitin ligase RNF10 modifies 40S ribosomal subunits of ribosomes compromised in translation.
The characterization of distinct classes of misfolded proteins uncovers differential effects in yeast compromised for proteasome function.
Affinity proteomics and deglycoproteomics uncover novel EDEM2 endogenous substrates and an integrative ERAD network.
Activation of salt Inducible Kinases, IRE1 and PERK leads to Sec bodies formation in Drosophila S2 cells.
A general role for MIA3/TANGO1 in secretory pathway organization and function.
DYT-TOR1A subcellular proteomics reveals selective vulnerability of the nuclear proteome to cell stress.
Unfolded protein response inhibitors cure group A streptococcal necrotizing fasciitis by modulating host asparagine.
Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway.
Proteolytic processing of secretory pathway kinase Fam20C by site-1 protease promotes biomineralization.
Exploring the Role of Endoplasmic Reticulum Stress in Hepatocellular Carcinoma through mining of the Human Protein Atlas.
Molecular functions of autophagy adaptors upon ubiquitin-driven mitophagy.
The stressosome, a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner.
The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes.
GRB2 enforces homology-directed repair initiation by MRE11.
Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol.
Human Multisubunit E3 Ubiquitin Ligase Required for Heterotrimeric G-Protein β-Subunit Ubiquitination and Downstream Signaling.

EMBO Rep. 2021 Aug 02. e52289

Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy, and Collagen quality control.

Alessio Reggio, Viviana Buonomo, Rayene Berkane, Ramachandra M Bhaskara, Mariana Tellechea, Ivana Peluso, Elena Polishchuk, Giorgia Di Lorenzo, Carmine Cirillo, Marianna Esposito, Adeela Hussain, Antje K Huebner, Christian A Hübner, Carmine Settembre, Gerhard Hummer, Paolo Grumati, Alexandra Stolz.

  Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways.

Keywords:  Collagen; ER stress; ER-phagy; FAM134; autophagy

DOI:  https://doi.org/10.15252/embr.202052289
Cell Death Discov. 2021 Aug 05. 7(1): 203

Dual topology of co-chaperones at the membrane of the endoplasmic reticulum.

Lea Daverkausen-Fischer, Felicitas Pröls.

Dual topologies of proteins at the ER membrane are known for a variety of proteins allowing the same protein to exert different functions according to the topology adopted. A dual topology of the co-chaperone ERdj4, which resides in the endoplasmic reticulum (ER), was proposed recently, a thesis that we found to align all published data and existing controversies into one whole picture. The aim of this review is to reassess all primary data available in the literature on ER-resident Hsp40 co-chaperones with respect to their topology. After careful and critical analyses of all experimental data published so far, we identified, next to ERdj4, two other co-chaperones, ERdj3 and ERdj6, that also display features of a dual topology at the ER membrane. We assume that during cellular stress subpools of some ER-resident J protein can alter their topology so that these proteins can exert different functions in order to adapt to cellular stress.

DOI: https://doi.org/10.1038/s41420-021-00594-x
Proc Natl Acad Sci U S A. 2021 Aug 10. pii: e2105324118. [Epub ahead of print]118(32):

The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells.

Jean-Baptiste Luizet, Julie Raymond, Thais Lourdes Santos Lacerda, Emeline Barbieux, Stanimir Kambarev, Magali Bonici, Frédérique Lembo, Kévin Willemart, Jean-Paul Borg, Jean Celli, Francine C A Gérard, Eric Muraille, Jean-Pierre Gorvel, Suzana P Salcedo.

  Perturbation of the endoplasmic reticulum (ER), a central organelle of the cell, can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control ensures that cells can respond and adapt to stress via the unfolded protein response (UPR) and that only correctly assembled proteins reach their destination. Interestingly, several bacterial pathogens hijack the ER to establish an infection. However, it remains poorly understood how bacterial pathogens exploit ER quality-control functions to complete their intracellular cycle. Brucella spp. replicate extensively within an ER-derived niche, which evolves into specialized vacuoles suited for exit from infected cells. Here we present Brucella-secreted protein L (BspL), a Brucella abortus effector that interacts with Herp, a central component of the ER-associated degradation (ERAD) machinery. We found that BspL enhances ERAD at the late stages of the infection. BspL targeting of Herp and ERAD allows tight control of the kinetics of autophagic Brucella-containing vacuole formation, delaying the last step of its intracellular cycle and cell-to-cell spread. This study highlights a mechanism by which a bacterial pathogen hijacks ERAD components for fine regulation of its intracellular trafficking.

Keywords:  Brucella; ERAD; Herp; trafficking

DOI:  https://doi.org/10.1073/pnas.2105324118
Autophagy. 2021 Aug 02. 1-13

STING1 is essential for an RNA-virus triggered autophagy.

Rui Zhang, Xiaodong Qin, Yang Yang, Xueliang Zhu, Shuaiyang Zhao, Zhixiong Zhang, Qianlong Su, Zhixun Zhao, Xiangping Yin, Xuelian Meng, Zhidong Zhang, Yanmin Li.

  While the functions of STING1 (stimulator of interferon response cGAMP interactor 1) during DNA virus infection had been well documented, the roles STING1 plays during RNA viruses infection is obscure. Infection with foot-and-mouth disease virus (FMDV), a well-known picornavirus, induces endoplasmic reticulum (ER) stress response and autophagy. Here, we found that the FMDV-induced integrated stress response originates from the cellular pattern recognition receptor DDX58/RIG-I (DExD/H-box helicase 58). DDX58 transmits signals to the ER-anchored adaptor protein STING1, which specifically activates the EIF2AK3/PERK (eukaryotic translation initiation factor 2A)-dependent integrated stress response and finally leads to reticulophagy and degradation of STING1 itself. Knockdown/knockout of STING1 or EIF2AK3 suppresses FMDV genome replication and viral protein expression. Reticulophagy induction by STING1 does not require its translocation to the Golgi or IFN response activation. However, STING1 polymerization is necessary for the FMDV-induced integrated stress response and reticulophagy. Our work illustrated the signaling cascades that mediate the cellular stress response to FMDV infection and indicated that induction of autophagy in response to both DNA and RNA virus infection may be an evolutionarily conserved function of STING1. Abbreviations: ATF6: activating transcription factor 6; CGAS: cyclic GMP-AMP synthase; DDX58/RIG-I: DExD/H-box helicase 58; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 2; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FMD: foot-and-mouth disease; FMDV: foot-and-mouth disease virus; IFIH1/MDA5: interferon induced with helicase C domain 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; RETREG1/FAM134B: reticulophagy regulator 1; STING1: stimulator of interferon response cGAMP interactor 1; TCID50: 50% tissue culture infectious dose; XBP1: X-box binding protein 1.

Keywords:  Autophagy; EIF2AK3; RNA virus; STING1; integrated stress response

DOI:  https://doi.org/10.1080/15548627.2021.1959086
Biochem Soc Trans. 2021 Aug 02. pii: BST20210496. [Epub ahead of print]

Are cachexia-associated tumors transmitTERS of ER stress?

Ana Sayuri Yamagata, Paula Paccielli Freire.

  Cancer cachexia is associated with deficient response to chemotherapy. On the other hand, the tumors of cachectic patients remarkably express more chemokines and have higher immune infiltration. For immunogenicity, a strong induction of the unfolded protein response (UPR) is necessary. UPR followed by cell surface exposure of calreticulin on the dying tumor cell is essential for its engulfment by macrophages and dendritic cells. However, some tumor cells upon endoplasmic reticulum (ER) stress can release factors that induce ER stress to other cells, in the so-called transmissible ER stress (TERS). The cells that received TERS produce more interleukin 6 (IL-6) and chemokines and acquire resistance to subsequent ER stress, nutrient deprivation, and genotoxic stress. Since ER stress enhances the release of extracellular vesicles (EVs), we suggest they can mediate TERS. It was found that ER stressed cachexia-inducing tumor cells transmit factors that trigger ER stress in other cells. Therefore, considering the role of EVs in cancer cachexia, the release of exosomes can possibly play a role in the process of blunting the immunogenicity of the cachexia-associated tumors. We propose that TERS can cause an inflammatory and immunosuppressive phenotype in cachexia-inducing tumors.

Keywords:  ER stress; cachexia; cancer; immunology; tumor microenvironment

DOI:  https://doi.org/10.1042/BST20210496
Front Plant Sci. 2021 ;12 707378

A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities.

Danish Diwan, Xiaoyu Liu, Caroline F Andrews, Karolina M Pajerowska-Mukhtar.

  The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro, the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.

Keywords:  Arabidopsis thaliana; ER stress; IRE1; Nicotiana benthamiana; RNase-dead; bZIP60; in vitro mRNA cleavage; unfolded protein response

DOI:  https://doi.org/10.3389/fpls.2021.707378
Cell Rep. 2021 Aug 03. pii: S2211-1247(21)00891-3. [Epub ahead of print]36(5): 109468

The E3 ubiquitin ligase RNF10 modifies 40S ribosomal subunits of ribosomes compromised in translation.

Aitor Garzia, Cindy Meyer, Thomas Tuschl.

  Reversible monoubiquitination of small subunit ribosomal proteins RPS2/uS5 and RPS3/uS3 has been noted to occur on ribosomes involved in ZNF598-dependent mRNA surveillance. Subsequent deubiquitination of RPS2 and RPS3 by USP10 is critical for recycling of stalled ribosomes in a process known as ribosome-associated quality control. Here, we identify and characterize the RPS2- and RPS3-specific E3 ligase Really Interesting New Gene (RING) finger protein 10 (RNF10) and its role in translation. Overexpression of RNF10 increases 40S ribosomal subunit degradation similarly to the knockout of USP10. Although a substantial fraction of RNF10-mediated RPS2 and RPS3 monoubiquitination results from ZNF598-dependent sensing of collided ribosomes, ZNF598-independent impairment of translation initiation and elongation also contributes to RPS2 and RPS3 monoubiquitination. RNF10 photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies crosslinked mRNAs, tRNAs, and 18S rRNAs, indicating recruitment of RNF10 to ribosomes stalled in translation. These impeded ribosomes are tagged by ubiquitin at their 40S subunit for subsequent programmed degradation unless rescued by USP10.

Keywords:  E3 ubiquitin ligase; RNA-binding protein; mRNA surveillance; regulatory protein ubiquitination; ribosome collision; ribosome-associated quality control; translation

DOI:  https://doi.org/10.1016/j.celrep.2021.109468
FEBS Lett. 2021 Aug 06.

The characterization of distinct classes of misfolded proteins uncovers differential effects in yeast compromised for proteasome function.

Grace D Burns, Olivia E Hilal, Zhihao Sun, Karl-Richard Reutter, G Michael Preston, Andrew A Augustine, Jeffrey L Brodsky, Christopher J Guerriero.

Maintenance of the proteome (proteostasis) is essential for cellular homeostasis and prevents cytotoxic stress responses that arise from protein misfolding. However, little is known about how different types of misfolded proteins impact homeostasis, especially when protein degradation pathways are compromised. We examined the effects of misfolded protein expression on yeast growth by characterizing a suite of substrates possessing the same aggregation-prone domain but engaging different quality control pathways. We discovered that treatment with a proteasome inhibitor was more toxic in yeast expressing misfolded membrane proteins, and this growth defect was mirrored in yeast lacking a proteasome-specific transcription factor, Rpn4p. These results highlight weaknesses in the proteostasis network's ability to handle the stress arising from an accumulation of misfolded membrane proteins.

DOI: https://doi.org/10.1002/1873-3468.14172
Mol Cell Proteomics. 2021 Jul 28. pii: S1535-9476(21)00097-9. [Epub ahead of print] 100125

Affinity proteomics and deglycoproteomics uncover novel EDEM2 endogenous substrates and an integrative ERAD network.

Cristian V A Munteanu, Gabriela N Chirițoiu, Marioara Chirițoiu, Simona Ghenea, Andrei-Jose Petrescu, Ștefana M Petrescu.

  Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system (ERQC) for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2 associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco-clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosites analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins we used affinity-proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ERQC and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, SILAC-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including ITGA1 and PCDH2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whilst ITGA1 and PCDH2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of these glycoproteins degradation and trafficking. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.

Keywords:  EDEM2; ER quality control; ERAD; glycoproteomics; mass spectrometry; melanoma; proteomics; pulse-SILAC

DOI:  https://doi.org/10.1016/j.mcpro.2021.100125
J Cell Sci. 2021 Aug 05. pii: jcs.258685. [Epub ahead of print]

Activation of salt Inducible Kinases, IRE1 and PERK leads to Sec bodies formation in Drosophila S2 cells.

Chujun Zhang, Wessel van Leeuwen, Marloes Blotenburg, Angelica Aguilera-Gomez, Sem Brussee, Rianne Grond, Harm H Kampinga, Catherine Rabouille.

  The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the ER exit sites under the stress of amino-acid starvation. Here we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt inducible kinases (SIK) by Na+ stress, that when it is strong is sufficient. The second is activation of IRE1 and PERK downstream of ER stress induced by absence of amino- acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK and IRE1/PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work pioneers the role of SIK in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino-acids and salt.

Keywords:  Amino-acid starvation; Drosophila S2 cells; Phase separation; Salt stress; Sec body; Unfolded Protein Response

DOI:  https://doi.org/10.1242/jcs.258685
J Cell Sci. 2021 Aug 05. pii: jcs.259075. [Epub ahead of print]

A general role for MIA3/TANGO1 in secretory pathway organization and function.

Janine McCaughey, Nicola L Stevenson, Judith M Mantell, Chris R Neal, Alex Paterson, Kate Heesom, David J Stephens.

  Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum, an essential process in eukaryotic cells. In vertebrates, the Mia3 gene encodes two major forms of Transport ANd Golgi Organization Protein 1 (TANGO1S and TANGO1L), previously implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics, and proteomics we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function including limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 and SURF4 to the ER-Golgi Intermediate Compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo including small soluble proteins. Our data support a general role for Mia3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells.

Keywords:  COPII; Collagen; ERGIC; Golgi; Secretory pathway; TANGO1

DOI:  https://doi.org/10.1242/jcs.259075
Neurobiol Dis. 2021 Aug 03. pii: S0969-9961(21)00213-8. [Epub ahead of print] 105464

DYT-TOR1A subcellular proteomics reveals selective vulnerability of the nuclear proteome to cell stress.

Kunal Shroff, Zachary F Caffall, Nicole Calakos.

  TorsinA is a AAA+ ATPase that shuttles between the ER lumen and outer nuclear envelope in an ATP-dependent manner and is functionally implicated in nucleocytoplasmic transport. We hypothesized that the DYT-TOR1A dystonia disease-causing variant, ΔE TorsinA, may therefore disrupt the normal subcellular distribution of proteins between the nuclear and cytosolic compartments. To test this hypothesis, we performed proteomic analysis on nuclear and cytosolic subcellular fractions from DYT-TOR1A and wildtype mouse embryonic fibroblasts (MEFs). We further examined the compartmental proteomes following exposure to thapsigargin (Tg), an endoplasmic reticulum (ER) stressor, because DYT-TOR1A dystonia models have previously shown abnormalities in cellular stress responses. Across both subcellular compartments, proteomes of DYT-TOR1A cells showed basal state disruptions consistent with an activated stress response, and in response to thapsigargin, a blunted stress response. However, the DYT-TOR1A nuclear proteome under Tg cell stress showed the most pronounced and disproportionate degree of protein disruptions - 3-fold greater than all other conditions. The affected proteins extended beyond those typically associated with stress responses, including enrichments for processes critical for neuronal synaptic function. These findings highlight the advantage of subcellular proteomics to reveal events that localize to discrete subcellular compartments and refine thinking about the mechanisms and significance of cell stress in DYT-TOR1A pathogenesis.

Keywords:  Compartment-specific proteome; Dystonia; Movement disorder; Stress response; Subcellular fractionation; TorsinA

DOI:  https://doi.org/10.1016/j.nbd.2021.105464
Sci Transl Med. 2021 Aug 04. pii: eabd7465. [Epub ahead of print]13(605):

Unfolded protein response inhibitors cure group A streptococcal necrotizing fasciitis by modulating host asparagine.

Aparna Anand, Abhinay Sharma, Miriam Ravins, Debabrata Biswas, Poornima Ambalavanan, Kimberly Xuan Zhen Lim, Rachel Ying Min Tan, Atul Kumar Johri, Boaz Tirosh, Emanuel Hanski.

Group A streptococcus (GAS) is among the top 10 causes of mortality from an infectious disease, producing mild to invasive life-threatening manifestations. Necrotizing fasciitis (NF) is characterized by a rapid GAS spread into fascial planes followed by extensive tissue destruction. Despite prompt treatments of antibiotic administration and tissue debridement, mortality from NF is still high. Moreover, there is no effective vaccine against GAS, and early diagnosis of NF is problematic because its clinical presentations are not specific. Thus, there is a genuine need for effective treatments against GAS NF. Previously, we reported that GAS induces endoplasmic reticulum (ER) stress to gain asparagine from the host. Here, we demonstrate that GAS-mediated asparagine induction and release occur through the PERK-eIF2α-ATF4 branch of the unfolded protein response. Inhibitors of PERK or integrated stress response (ISR) blocked the formation and release of asparagine by infected mammalian cells, and exogenously added asparagine overcame this inhibition. Moreover, in a murine model of NF, we show that the inhibitors minimized mortality when mice were challenged with a lethal dose of GAS and reduced bacterial counts and lesion size when mice were challenged with a sublethal dose. Immunohistopathology studies demonstrated that PERK/ISR inhibitors protected mice by enabling neutrophil infiltration into GAS-infected fascia and reducing the pro-inflammatory response that causes tissue damage. Inhibitor treatment was also effective in mice when started at 12 hours after infection. We conclude that host metabolic alteration induced by PERK or ISR inhibitors is a promising therapeutic strategy to treat highly invasive GAS infections.

DOI: https://doi.org/10.1126/scitranslmed.abd7465
EMBO J. 2021 Aug 04. e107413

Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway.

Julio C Y Liu, Ulrike Kühbacher, Nicolai B Larsen, Nikoline Borgermann, Dimitriya H Garvanska, Ivo A Hendriks, Leena Ackermann, Peter Haahr, Irene Gallina, Claire Guérillon, Emma Branigan, Ronald T Hay, Yoshiaki Azuma, Michael Lund Nielsen, Julien P Duxin, Niels Mailand.

  DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.

Keywords:  DNA repair; DNA-protein crosslinks; SUMO; genome stability; ubiquitin

DOI:  https://doi.org/10.15252/embj.2020107413
Proc Natl Acad Sci U S A. 2021 Aug 10. pii: e2100133118. [Epub ahead of print]118(32):

Proteolytic processing of secretory pathway kinase Fam20C by site-1 protease promotes biomineralization.

Xinxin Chen, Jianchao Zhang, Pulan Liu, Yangyang Wei, Xi'e Wang, Junyu Xiao, Chih-Chen Wang, Lei Wang.

  Family with sequence similarity 20C (Fam20C), the major protein kinase in the secretory pathway, generates the vast majority of the secreted phosphoproteome. However, the regulatory mechanisms of Fam20C transport, secretion, and function remain largely unexplored. Here, we show that Fam20C exists as a type II transmembrane protein within the secretory compartments, with its N-terminal signal peptide-like region serving as a membrane anchor for Golgi retention. The secretion and kinase activity of Fam20C are governed by site-1 protease (S1P), a key regulator of cholesterol homeostasis. We find that only mature Fam20C processed by S1P functions in osteoblast differentiation and mineralization. Together, our findings reveal a unique mechanism for Fam20C secretion and activation via proteolytic regulation, providing a molecular link between biomineralization and lipid metabolism.

Keywords:  Fam20C; Golgi; phosphorylation; proteolysis; site-1 protease

DOI:  https://doi.org/10.1073/pnas.2100133118
Biology (Basel). 2021 Jul 09. pii: 640. [Epub ahead of print]10(7):

Exploring the Role of Endoplasmic Reticulum Stress in Hepatocellular Carcinoma through mining of the Human Protein Atlas.

Nataša Pavlović, Femke Heindryckx.

  Endoplasmic reticulum (ER) stress and actors of unfolded protein response (UPR) have emerged as key hallmarks of hepatocarcinogenesis. Numerous reports have shown that the main actors in the UPR pathways are upregulated in HCC and contribute to the different facets of tumor initiation and disease progression. Furthermore, ER-stress inducers and inhibitors have shown success in preclinical HCC models. Despite the mounting evidence of the UPR's involvement in HCC pathogenesis, it remains unclear how ER-stress components can be used safely and effectively as therapeutic targets or predictive biomarkers for HCC patients. In an effort to add a clinical context to these findings and explore the translational potential of ER-stress in HCC, we performed a systematic overview of UPR-associated proteins as predictive biomarkers in HCC by mining the Human Protein Atlas database. Aside from evaluating the prognostic value of these markers in HCC, we discussed their expression in relation to patient age, sex, ethnicity, disease stage, and tissue localization. We thereby identified 44 UPR-associated proteins as unfavorable prognostic markers in HCC. The expression of these markers was found to be higher in tumors compared to the stroma of the hepatic HCC patient tissues.

Keywords:  endoplasmic reticulum stress; hepatocellular carcinoma; unfolded protein response

DOI:  https://doi.org/10.3390/biology10070640
Biochim Biophys Acta Gen Subj. 2021 Jul 28. pii: S0304-4165(21)00131-8. [Epub ahead of print]1865(10): 129972

Molecular functions of autophagy adaptors upon ubiquitin-driven mitophagy.

Koji Yamano, Waka Kojima.

BACKGROUND: Perturbations in organellar health can lead to an accumulation of unwanted and/or damaged organelles that are toxic to the cell and which can contribute to the onset of neurodegenerative diseases such as Parkinson's disease. Mitochondrial health is particularly critical given the indispensable role the organelle has not only in adenosine triphosphate production but also other metabolic processes. Byproducts of oxidative respiration, such as reactive oxygen species, however, can negatively impact mitochondrial fitness. Consequently, selective degradation of damaged mitochondria, which occurs via a specific autophagic process termed mitophagy, is essential for normal cell maintenance.
SCOPE OF REVIEW: Recent accumulating evidence has shown that autophagy adaptors (also referred to as autophagy receptors) play critical roles in connecting ubiquitinated mitochondria with the autophagic machinery of the autophagy-lysosome pathway that is required for degradation. In this review, we focus on our current understanding of the autophagy adaptor mechanisms underlying PINK1/Parkin-driven mitophagy.
MAJOR CONCLUSIONS: Although autophagy adaptors are canonically defined as proteins that possess ubiquitin-binding domains and ATG8s-binding motifs, the recent identification of novel binding partners has contributed to the development of a more sophisticated model for how autophagy adaptors contribute to the molecular hub that organizes autophagic cargo-degradation.
GENERAL SIGNIFICANCE: Although mitophagy is recognized as one of the selective autophagy pathways that removes dysfunctional mitochondria, a more nuanced understanding of the interactions connecting autophagy adaptors and their associated proteins is needed to gain deeper insights into the fundamental biological processes underlying human diseases, including neurodegenerative disorders. This review is part of a Special Issue entitled Mitophagy.

DOI: https://doi.org/10.1016/j.bbagen.2021.129972
J Cell Mol Med. 2021 Aug 07.

The stressosome, a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner.

Katarzyna Mnich, Izabela Koryga, Karolina Pakos-Zebrucka, Melissa Thomas, Susan E Logue, Leif A Eriksson, Adrienne M Gorman, Afshin Samali.

  Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the 'stressosome'. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1β-converting enzyme-inhibitory protein (cFLIPL ), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.

Keywords:  apoptosis; autophagy; caspase; cell stress; integrated stress response

DOI:  https://doi.org/10.1111/jcmm.16840
Proc Natl Acad Sci U S A. 2021 Aug 10. pii: e2013500118. [Epub ahead of print]118(32):

The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes.

Kishu Ranjan, Matija Hedl, Clara Abraham.

  Balancing microbial-induced cytokines and microbial clearance is critical at mucosal sites such as the intestine. How the inflammatory bowel disease (IBD)-associated gene RNF186 regulates this balance is unclear. We found that macrophages from IBD-risk rs6426833 carriers in the RNF186 region showed reduced cytokines to stimulation through multiple pattern recognition receptors (PRRs). Upon stimulation of PRRs, the E3-ubiquitin ligase RNF186 promoted ubiquitination of signaling complex molecules shared across PRRs and those unique to select PRRs. Furthermore, RNF186 was required for PRR-initiated signaling complex assembly and downstream signaling. RNF186, along with its intact E3-ubiquitin ligase activity, was required for optimal PRR-induced antimicrobial reactive oxygen species, reactive nitrogen species, and autophagy pathways and intracellular bacterial clearance in human macrophages and for bacterial clearance in intestinal myeloid cells. Cells transfected with the rare RNF186-A64T IBD-risk variant and macrophages from common rs6426833 RNF186 IBD-risk carriers demonstrated a reduction in these RNF186-dependent outcomes. These studies identify mechanisms through which RNF186 regulates innate immunity and show that RNF186 IBD-risk variants demonstrate a loss of function in PRR-initiated outcomes.

Keywords:  inflammatory bowel disease; innate immunity; macrophage; ubiquitination

DOI:  https://doi.org/10.1073/pnas.2013500118
Sci Adv. 2021 Aug;pii: eabe9254. [Epub ahead of print]7(32):

GRB2 enforces homology-directed repair initiation by MRE11.

Zu Ye, Shengfeng Xu, Yin Shi, Albino Bacolla, Aleem Syed, Davide Moiani, Chi-Lin Tsai, Qiang Shen, Guang Peng, Paul G Leonard, Darin E Jones, Bin Wang, John A Tainer, Zamal Ahmed.

DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.

DOI: https://doi.org/10.1126/sciadv.abe9254
Cell Death Differ. 2021 Aug 05.

Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol.

Christian T Hellwig, M Eugenia Delgado, Josip Skoko, Lydia Dyck, Carol Hanna, Alexa Wentges, Claudia Langlais, Cathrin Hagenlocher, Alexandra Mack, David Dinsdale, Kelvin Cain, Marion MacFarlane, Markus Rehm.

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.

DOI: https://doi.org/10.1038/s41418-021-00843-7
J Proteome Res. 2021 Aug 03.

Human Multisubunit E3 Ubiquitin Ligase Required for Heterotrimeric G-Protein β-Subunit Ubiquitination and Downstream Signaling.

Brian D Young, Jihui Sha, Ajay A Vashisht, James A Wohlschlegel.

  G-protein-coupled receptors (GPCRs) initiate intracellular signaling events through heterotrimeric G-protein α-subunits (Gα) and the βγ-subunit dimer (Gβγ). In this study, we utilized mass spectrometry to identify novel regulators of Gβγ signaling in human cells. This prompted our characterization of KCTD2 and KCTD5, two related potassium channel tetramerization domain (KCTD) proteins that specifically recognize Gβγ. We demonstrated that these KCTD proteins are substrate adaptors for a multisubunit CUL3-RING ubiquitin ligase, in which a KCTD2-KCTD5 hetero-oligomer associates with CUL3 through KCTD5 subunits and recruits Gβγ through both KCTD proteins in response to G-protein activation. These KCTD proteins promote monoubiquitination of lysine-23 within Gβ1/2 in vitro and in HEK-293 cells. Depletion of these adaptors from cancer cell lines sharply impairs downstream signaling. Together, our studies suggest that a KCTD2-KCTD5-CUL3-RING E3 ligase recruits Gβγ in response to signaling, monoubiquitinates lysine-23 within Gβ1/2, and regulates Gβγ effectors to modulate downstream signal transduction.

Keywords:  KCTD proteins; cAMP signaling; cullin-RING E3 ubiquitin ligase; heterotrimeric G-proteins; ubiquitination

DOI:  https://doi.org/10.1021/acs.jproteome.1c00292