bims-proteo 2023-07-23 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–07–23
23 papers selected by
Eric Chevet, INSERM

ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis.
Orphan quality control shapes network dynamics and gene expression.
Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery.
Genetic inactivation of essential HSF1 reveals an isolated transcriptional stress response selectively induced by protein misfolding.
Stranger Twins: A Tale of Resemblance and Contrast Between VAP Proteins.
The C-terminal tail of CSNAP attenuates the CSN complex.
Analysis of ATG4C function in vivo.
Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome.
IL-1β turnover by the UBE2L3 ubiquitin conjugating enzyme and HECT E3 ligases limits inflammation.
A signal peptide peptidase is required for ER-symbiosome proximal association and protein secretion.
UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability.
Homeostatic regulation of ribosomal proteins by ubiquitin-independent cotranslational degradation.
A spatially defined human Notch receptor interaction network reveals Notch intracellular storage and Ataxin-2-mediated fast recycling.
Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape.
Unravelling the role of NFE2L1 in stress responses and related diseases.
Bridging Energy Need and Feeding Behavior: The Impact of eIF2α Phosphorylation in AgRP Neurons.
Parkin regulates amino acid homeostasis at mitochondria-lysosome (M/L) contact sites in Parkinson's disease.
Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome.
Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Neutrophilic Airway Inflammation.
Design of Cell-Permeable Inhibitors of Eukaryotic Translation Initiation Factor 4E (eIF4E) for Inhibiting Aberrant Cap-Dependent Translation in Cancer.
Mega-scale experimental analysis of protein folding stability in biology and design.
Autophagy orchestrates the crosstalk between cells and organs.
Phase separated ribosome-nascent chain complexes in genotoxic stress response.

J Cell Biol. 2023 Sep 04. pii: e202208060. [Epub ahead of print]222(9):

ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis.

Ioanna Myronidi, Andreas Ring, Fei Wu, Per O Ljungdahl.

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane-localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone-substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3-AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3-AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes.

DOI: https://doi.org/10.1083/jcb.202208060
Cell. 2023 Jul 14. pii: S0092-8674(23)00691-8. [Epub ahead of print]

Orphan quality control shapes network dynamics and gene expression.

Kevin G Mark, SriDurgaDevi Kolla, Jacob D Aguirre, Danielle M Garshott, Stefan Schmitt, Diane L Haakonsen, Christina Xu, Lukas Kater, Georg Kempf, Brenda Martínez-González, David Akopian, Stephanie K See, Nicolas H Thomä, Michael Rapé.

  All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.

Keywords:  MCRS1; MYC; UBR5; branched ubiquitin chain; orphan quality control; proteasome; stem cell; transcription factor; ubiquitin

DOI:  https://doi.org/10.1016/j.cell.2023.06.015
Cell Rep. 2023 Jul 13. pii: S2211-1247(23)00818-5. [Epub ahead of print]42(7): 112807

Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery.

Sarah J Backe, Rebecca A Sager, Jennifer A Heritz, Laura A Wengert, Katherine A Meluni, Xavier Aran-Guiu, Barry Panaretou, Mark R Woodford, Chrisostomos Prodromou, Dimitra Bourboulia, Mehdi Mollapour.

  Cellular homeostasis relies on both the chaperoning of proteins and the intracellular degradation system that delivers cytoplasmic constituents to the lysosome, a process known as autophagy. The crosstalk between these processes and their underlying regulatory mechanisms is poorly understood. Here, we show that the molecular chaperone heat shock protein 90 (Hsp90) forms a complex with the autophagy-initiating kinase Atg1 (yeast)/Ulk1 (mammalian), which suppresses its kinase activity. Conversely, environmental cues lead to Atg1/Ulk1-mediated phosphorylation of a conserved serine in the amino domain of Hsp90, inhibiting its ATPase activity and altering the chaperone dynamics. These events impact a conformotypic peptide adjacent to the activation and catalytic loop of Atg1/Ulk1. Finally, Atg1/Ulk1-mediated phosphorylation of Hsp90 leads to dissociation of the Hsp90:Atg1/Ulk1 complex and activation of Atg1/Ulk1, which is essential for initiation of autophagy. Our work indicates a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase Atg1/Ulk1 and consequent maintenance of cellular proteostasis.

Keywords:  CP: Molecular biology; Hsp90; Ulk1; atg1; autophagy; chaperone; chaperone code; co-chaperone; phosphorylation

DOI:  https://doi.org/10.1016/j.celrep.2023.112807
Mol Biol Cell. 2023 Jul 19. mbcE23050153

Genetic inactivation of essential HSF1 reveals an isolated transcriptional stress response selectively induced by protein misfolding.

Michela Ciccarelli, Anna E Masser, Jayasankar Mohanakrishnan Kaimal, Jordi Planells, Claes Andréasson.

Heat Shock Factor 1 (Hsf1) in yeast drives the basal transcription of key proteostasis factors and its activity is induced as part of the core heat shock response. Exploring Hsf1 specific functions has been challenging due to the essential nature of the HSF1 gene and the extensive overlap of target promoters with environmental stress response (ESR) transcription factors Msn2 and Msn4 (Msn2/4). In this study, we constructed a viable hsf1∆ strain by replacing the HSF1 open reading frame with genes that constitutively express Hsp40, Hsp70 and Hsp90 from Hsf1-independent promoters. Phenotypic analysis showed that the hsf1∆ strain grow slowly, is sensitive to heat as well as protein misfolding and accumulates protein aggregates. Transcriptome analysis revealed that the transcriptional response to protein misfolding induced by azetidine-2-carboxylic acid is fully dependent of Hsf1. In contrast, the hsf1∆ strain responded to heat shock through the ESR. Following HS, Hsf1 and Msn2/4 showed functional compensatory induction with stronger activation of the remaining stress pathway when the other branch was inactivated. Thus, we provide a long overdue genetic test of the function of Hsf1 in yeast using the novel hsf1∆ construct. Our data highlight that the accumulation of misfolded proteins is uniquely sensed by Hsf1-Hsp70 chaperone titration inducing a highly selective transcriptional stress response.

DOI: https://doi.org/10.1091/mbc.E23-05-0153
Contact (Thousand Oaks). 2023 Jan-Dec;6:6 25152564231183897

Stranger Twins: A Tale of Resemblance and Contrast Between VAP Proteins.

Mélody Subra, Zoé Grimanelli, Romain Gautier, Bruno Mesmin.

  When considering the vesicle-associated membrane protein-associated protein (VAP) family, major receptors at the surface of the endoplasmic reticulum (ER), it appears that VAP-A and VAP-B paralogs largely overlap in structure and function, and that specific features to distinguish these two proteins hardly exist or are poorly documented. Here, we question the degree of redundancy between VAP-A and VAP-B: is one simply a backup plan, in case of loss of function of one of the two genes, or are there molecular and functional divergences that would explain their maintenance during evolution?

Keywords:  VAP; endoplasmic reticulum; membrane contact sites; structural biology; tether

DOI:  https://doi.org/10.1177/25152564231183897
Life Sci Alliance. 2023 Oct;pii: e202201634. [Epub ahead of print]6(10):

The C-terminal tail of CSNAP attenuates the CSN complex.

Maria G Füzesi-Levi, Gili Ben-Nissan, Dina Listov, Yael Fridmann Sirkis, Zvi Hayouka, Sarel Fleishman, Michal Sharon.

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitination is the cullin-RING ligases (CRLs). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumor progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises ∼300 complexes, all of which are regulated by the COP9 signalosome complex (CSN). Therefore, CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN-interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.

DOI: https://doi.org/10.26508/lsa.202201634
Autophagy. 2023 Jul 17. 1-22

Analysis of ATG4C function in vivo.

Isaac Tamargo-Gómez, Gemma G Martínez-García, María F Suárez, Pablo Mayoral, Gabriel Bretones, Aurora Astudillo, Jesús Prieto-Lloret, Christina Sveen, Antonio Fueyo, Nikolai Engedal, Carlos López-Otín, Guillermo Mariño.

   ABBREVIATIONS: ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).

Keywords:  Animal models; GABARAP; LC3; autophagy; fibrosarcoma; lymphocyte

DOI:  https://doi.org/10.1080/15548627.2023.2234799
Neuron. 2023 Jul 13. pii: S0896-6273(23)00476-2. [Epub ahead of print]

Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome.

Mehdi Hooshmandi, Vijendra Sharma, Carolina Thörn Perez, Rapita Sood, Konstanze Krimbacher, Calvin Wong, Kevin C Lister, Alba Ureña Guzmán, Trevor D Bartley, Cecilia Rocha, Gilles Maussion, Emma Nadler, Patricia Margarita Roque, Ilse Gantois, Jelena Popic, Maxime Lévesque, Randal J Kaufman, Massimo Avoli, Elisenda Sanz, Karim Nader, Randi Jenssen Hagerman, Thomas M Durcan, Mauro Costa-Mattioli, Masha Prager-Khoutorsky, Jean-Claude Lacaille, Veronica Martinez-Cerdeno, Jay R Gibson, Kimberly M Huber, Nahum Sonenberg, Christos G Gkogkas, Arkady Khoutorsky.

  Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

Keywords:  autism; fragile X syndrome; integrated stress response; mRNA translation

DOI:  https://doi.org/10.1016/j.neuron.2023.06.017
Nat Commun. 2023 Jul 20. 14(1): 4385

IL-1β turnover by the UBE2L3 ubiquitin conjugating enzyme and HECT E3 ligases limits inflammation.

Vishwas Mishra, Anna Crespo-Puig, Callum McCarthy, Tereza Masonou, Izabela Glegola-Madejska, Alice Dejoux, Gabriella Dow, Matthew J G Eldridge, Luciano H Marinelli, Meihan Meng, Shijie Wang, Daniel J Bennison, Rebecca Morrison, Avinash R Shenoy.

The cytokine interleukin-1β (IL-1β) has pivotal roles in antimicrobial immunity, but also incites inflammatory disease. Bioactive IL-1β is released following proteolytic maturation of the pro-IL-1β precursor by caspase-1. UBE2L3, a ubiquitin conjugating enzyme, promotes pro-IL-1β ubiquitylation and proteasomal disposal. However, actions of UBE2L3 in vivo and its ubiquitin ligase partners in this process are unknown. Here we report that deletion of Ube2l3 in mice reduces pro-IL-1β turnover in macrophages, leading to excessive mature IL-1β production, neutrophilic inflammation and disease following inflammasome activation. An unbiased RNAi screen identified TRIP12 and AREL1 E3 ligases of the Homologous to E6 C-terminus (HECT) family in adding destabilising K27-, K29- and K33- poly-ubiquitin chains on pro-IL-1β. We show that precursor abundance determines mature IL-1β production, and UBE2L3, TRIP12 and AREL1 limit inflammation by shrinking the cellular pool of pro-IL-1β. Our study uncovers fundamental processes governing IL-1β homeostasis and provides molecular insights that could be exploited to mitigate its adverse actions in disease.

DOI: https://doi.org/10.1038/s41467-023-40054-x
Nat Commun. 2023 07 19. 14(1): 4355

A signal peptide peptidase is required for ER-symbiosome proximal association and protein secretion.

Jian Yang, Niu Zhai, Yuhui Chen, Luying Wang, Rujin Chen, Huairong Pan.

During legume-rhizobia symbiosis, differentiation of the symbiosome (engulfed intracellular rhizobia) is necessary for successful nitrogen fixation. To control symbiosome differentiation, host cell subcellular components, e.g., ER (endoplasmic reticulum), must adapt robustly to ensure large-scale host protein secretion to the new organelle. However, the key components controlling the adaption of ER in nodule cells remain elusive. We report that Medicago BID1, a nodule-specific signal peptide peptidase (SPP), is central to ER structural dynamics and host protein secretion. In bid1, symbiosome differentiation is blocked. BID1 localizes specifically to the ER membrane and expresses exclusively in nodule cells with symbiosomes. In the wild type ER forms proximal association structures with symbiosomes, but not in bid1. Consequently, in bid1 excessive ER stress responses are induced and ER-to-symbiosome protein secretion is impaired. In summary, a nodule-specific SPP is necessary for ER-symbiosome proximal association, host protein secretion, and symbiosome differentiation.

DOI: https://doi.org/10.1038/s41467-023-40008-3
Mol Cell. 2023 Jul 12. pii: S1097-2765(23)00475-6. [Epub ahead of print]

UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability.

Jonathan M Tsai, Jacob D Aguirre, Yen-Der Li, Jared Brown, Vivian Focht, Lukas Kater, Georg Kempf, Brittany Sandoval, Stefan Schmitt, Justine C Rutter, Pius Galli, Colby R Sandate, Jevon A Cutler, Charles Zou, Katherine A Donovan, Ryan J Lumpkin, Simone Cavadini, Paul M C Park, Quinlan Sievers, Charlie Hatton, Elizabeth Ener, Brandon D Regalado, Micah T Sperling, Mikołaj Słabicki, Jeonghyeon Kim, Rebecca Zon, Zinan Zhang, Peter G Miller, Roger Belizaire, Adam S Sperling, Eric S Fischer, Rafael Irizarry, Scott A Armstrong, Nicolas H Thomä, Benjamin L Ebert.

  Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.

Keywords:  HECT-E3 ligases; nuclear receptors; protein degradation; structural biology; ubiquitin ligases

DOI:  https://doi.org/10.1016/j.molcel.2023.06.028
Proc Natl Acad Sci U S A. 2023 07 25. 120(30): e2306152120

Homeostatic regulation of ribosomal proteins by ubiquitin-independent cotranslational degradation.

Donghong Ju, Li Li, Youming Xie.

  Ribosomes are the workplace for protein biosynthesis. Protein production required for normal cell function is tightly linked to ribosome abundance. It is well known that ribosomal genes are actively transcribed and ribosomal messenger RNAs (mRNAs) are rapidly translated, and yet ribosomal proteins have relatively long half-lives. These observations raise questions as to how homeostasis of ribosomal proteins is controlled. Here, we show that ribosomal proteins, while posttranslationally stable, are subject to high-level cotranslational protein degradation (CTPD) except for those synthesized as ubiquitin (Ub) fusion precursors. The N-terminal Ub moiety protects fused ribosomal proteins from CTPD. We further demonstrate that cotranslational folding efficiency and expression level are two critical factors determining CTPD of ribosomal proteins. Different from canonical posttranslational degradation, we found that CTPD of all the ribosomal proteins tested in this study does not require prior ubiquitylation. This work provides insights into the regulation of ribosomal protein homeostasis and furthers our understanding of the mechanism and biological significance of CTPD.

Keywords:  cotranslational protein degradation; proteasomal degradation; protein degradation; ribosomal proteins; ubiquitin-independent protein degradation

DOI:  https://doi.org/10.1073/pnas.2306152120
Cell Rep. 2023 Jul 15. pii: S2211-1247(23)00830-6. [Epub ahead of print]42(7): 112819

A spatially defined human Notch receptor interaction network reveals Notch intracellular storage and Ataxin-2-mediated fast recycling.

Weixiang Bian, Hua Jiang, Luxia Yao, Wanyu Hao, Lianfeng Wu, Xu Li.

  The Notch signaling pathway controls cell growth, differentiation, and fate decisions. Dysregulation of Notch signaling has been linked to various human diseases. Notch receptor resides in multiple cellular compartments, and its translocation plays a central role in pathway activation. However, the spatial regulation of Notch receptor functions remains largely elusive. Using TurboID-based proximity labeling followed by affinity purification and mass spectrometry, we establish a spatially defined human Notch receptor interaction network. Notch receptors interact with different proteins in distinct subcellular compartments to perform specific cellular functions. This spatially defined interaction network also reveals that a large fraction of NOTCH is stored at the endoplasmic reticulum (ER)-Golgi intermediate compartment and recruits Ataxin-2-dependent recycling machinery for rapid recycling, Notch signaling activation, and leukemogenesis. Our work provides insights into dynamic Notch receptor complexes with exquisite spatial resolution, which will help in elucidating the detailed regulation of Notch receptors and highlight potential therapeutic targets for Notch-related pathogenesis.

Keywords:  Ataxin-2; CP: Cell biology; ERGIC; Notch receptor interaction network; TurboID-based proximity labeling

DOI:  https://doi.org/10.1016/j.celrep.2023.112819
Cell Syst. 2023 Jul 19. pii: S2405-4712(23)00154-0. [Epub ahead of print]14(7): 551-562.e5

Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape.

Taivan Batjargal, Francesca Zappa, Ryan J Grant, Robert A Piscopio, Alex Chialastri, Siddharth S Dey, Diego Acosta-Alvear, Maxwell Z Wilson.

  The integrated stress response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered a photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics, we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR, which demonstrated the dependence of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords:  Cryptochrome 2; RNA-seq; cellular resilience; integrated stress response; optogenetics; stress memory landscape; systems biology

DOI:  https://doi.org/10.1016/j.cels.2023.06.001
Redox Biol. 2023 Jul 14. pii: S2213-2317(23)00220-3. [Epub ahead of print]65 102819

Unravelling the role of NFE2L1 in stress responses and related diseases.

Xingzhu Liu, Chang Xu, Wanglong Xiao, Nianlong Yan.

  The nuclear factor erythroid 2 (NF-E2)-related factor 1 (NFE2L1, also known as Nrf1) is a highly conserved transcription factor that belongs to the CNC-bZIP subfamily. Its significance lies in its control over redox balance, proteasome activity, and organ integrity. Stress responses encompass a series of compensatory adaptations utilized by cells and organisms to cope with extracellular or intracellular stress initiated by stressful stimuli. Recently, extensive evidence has demonstrated that NFE2L1 plays a crucial role in cellular stress adaptation by 1) responding to oxidative stress through the induction of antioxidative responses, and 2) addressing proteotoxic stress or endoplasmic reticulum (ER) stress by regulating the ubiquitin-proteasome system (UPS), unfolded protein response (UPR), and ER-associated degradation (ERAD). It is worth noting that NFE2L1 serves as a core factor in proteotoxic stress adaptation, which has been extensively studied in cancer and neurodegeneration associated with enhanced proteasomal stress. In these contexts, utilization of NFE2L1 inhibitors to attenuate proteasome "bounce-back" response holds tremendous potential for enhancing the efficacy of proteasome inhibitors. Additionally, abnormal stress adaptations of NFE2L1 and disturbances in redox and protein homeostasis contribute to the pathophysiological complications of cardiovascular diseases, inflammatory diseases, and autoimmune diseases. Therefore, a comprehensive exploration of the molecular basis of NFE2L1 and NFE2L1-mediated diseases related to stress responses would not only facilitate the identification of novel diagnostic and prognostic indicators but also enable the identification of specific therapeutic targets for NFE2L1-related diseases.

Keywords:  Adaptive responses; Cancer; NFE2L1; Neurodegeneration; Oxidative stress; Proteasome

DOI:  https://doi.org/10.1016/j.redox.2023.102819
Diabetes. 2023 Jul 21. pii: db230004. [Epub ahead of print]

Bridging Energy Need and Feeding Behavior: The Impact of eIF2α Phosphorylation in AgRP Neurons.

Kwang Kon Kim, Tae Hwan Lee, Byong Seo Park, Dasol Kang, Dong Hee Kim, Bora Jeong, Jin Woo Kim, Hye Rim Yang, Han Rae Kim, Sungho Jin, Sung Hoon Back, Jeong Woo Park, Jae Geun Kim, Byung Ju Lee.

Eukaryotic translation initiation factor 2α (eIF2α) is a key mediator of the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR). In mammals, eIF2α is phosphorylated by overnutrition-induced ER stress and is related to the development of obesity. Here, we studied the function of phosphorylated eIF2α (p-eIF2α) in AgRP neurons using a mouse model (AgRPeIF2αA/A) with an AgRP neuron-specific substitution from Ser 51 to Ala in eIF2α, which impairs eIF2α phosphorylation in AgRP neurons. These AgRPeIF2αA/A mice showed decreases in starvation-induced AgRP neuronal activity and food intake and also revealed an increased responsiveness to leptin. Intriguingly, impairment of eIF2α phosphorylation produced decreases in the starvation-induced expression of UPR and autophagy genes in AgRP neurons. Collectively, these findings suggest that eIF2α phosphorylation regulates AgRP neuronal activity by affecting intracellular responses such as the UPR and autophagy during starvation, thereby participating in the homeostatic control of wholebody energy metabolism.

DOI: https://doi.org/10.2337/db23-0004
Sci Adv. 2023 Jul 21. 9(29): eadh3347

Parkin regulates amino acid homeostasis at mitochondria-lysosome (M/L) contact sites in Parkinson's disease.

Wesley Peng, Leonie F Schröder, Pingping Song, Yvette C Wong, Dimitri Krainc.

Mutations in the E3 ubiquitin ligase parkin are the most common cause of early-onset Parkinson's disease (PD). Although parkin modulates mitochondrial and endolysosomal homeostasis during cellular stress, whether parkin regulates mitochondrial and lysosomal cross-talk under physiologic conditions remains unresolved. Using transcriptomics, metabolomics and super-resolution microscopy, we identify amino acid metabolism as a disrupted pathway in iPSC-derived dopaminergic neurons from patients with parkin PD. Compared to isogenic controls, parkin mutant neurons exhibit decreased mitochondria-lysosome contacts via destabilization of active Rab7. Subcellular metabolomics in parkin mutant neurons reveals amino acid accumulation in lysosomes and their deficiency in mitochondria. Knockdown of the Rab7 GTPase-activating protein TBC1D15 restores mitochondria-lysosome tethering and ameliorates cellular and subcellular amino acid profiles in parkin mutant neurons. Our data thus uncover a function of parkin in promoting mitochondrial and lysosomal amino acid homeostasis through stabilization of mitochondria-lysosome contacts and suggest that modulation of interorganelle contacts may serve as a potential target for ameliorating amino acid dyshomeostasis in disease.

DOI: https://doi.org/10.1126/sciadv.adh3347
Cell Rep. 2023 Jul 19. pii: S2211-1247(23)00828-8. [Epub ahead of print]42(8): 112817

Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome.

Jinli Ge, Ying Wang, Xindi Chen, Kaiwen Yu, Zhao-Qing Luo, Xiaoyun Liu, Jiazhang Qiu.

  Xenophagy is an evolutionarily conserved host defensive mechanism to eliminate invading microorganisms through autophagic machinery. The intracellular bacterial pathogen Legionella pneumophila can avoid clearance by the xenophagy pathway via the actions of multiple Dot/Icm effector proteins. Previous studies have shown that p62, an adaptor protein involved in xenophagy signaling, is excluded from Legionella-containing vacuoles (LCVs). Such defects are attributed to the multifunctional SidE family effectors (SidEs) that exhibit classic deubiquitinase (DUB) and phosphoribosyl ubiquitination (PR-ubiquitination) activities, yet the mechanism remains elusive. In the present study, we demonstrate that the host DUB USP14 is PR-ubiquitinated by SidEs at multiple serine residues, which impairs its DUB activity and its interactions with p62. The exclusion of p62 from the bacterial phagosome requires the ubiquitin ligase but not the DUB activity of SidEs. These results reveal that PR-ubiquitination of USP14 by SidEs contributes to the evasion of xenophagic clearance by L. pneumophila.

Keywords:  CP: Microbiology; Legionella; SdeA; SidEs; USP14; autophagy; effector proteins; p62; phosphoribosyl-linked serine ubiquitination; type IV secretion system; xenophagy

DOI:  https://doi.org/10.1016/j.celrep.2023.112817
bioRxiv. 2023 Jul 08. pii: 2023.06.30.547286. [Epub ahead of print]

Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Neutrophilic Airway Inflammation.

Dandan Wu, Xing Zhang, Kourtney M Zimmerly, Ruoning Wang, Amanda Livingston, Takao Iwawaki, Ashok Kumar, Xiang Wu, Michael A Mandell, Meilian Liu, Xuexian O Yang.

Treatment-refractory severe asthma manifests a neutrophilic phenotype associated with TH17 responses. Heightened unfolded protein responses (UPRs) are associated with the risk of asthma, including severe asthma. However, how UPRs participate in the deregulation of TH17 cells leading to this type of asthma remains elusive. In this study, we investigated the role of the UPR sensor IRE1 in TH17 cell function and neutrophilic airway inflammation. We found that IRE1 is induced in fungal asthma and is highly expressed in TH17 cells relative to naïve CD4 + T cells. Cytokine (e.g. IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by TH17 cells. Ern1 (encoding IRE1)-deficiency decreases the expression of ER stress factors and impairs the differentiation and cytokine secretion of TH17 cells. Genetic ablation of Ern1 leads to alleviated TH17 responses and airway neutrophilia in a Candida albicans asthma model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway in vivo and enhances TH17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPR- mediated secretory function of TH17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in TH17-biased TH2-low asthma.

DOI: https://doi.org/10.1101/2023.06.30.547286
J Med Chem. 2023 Jul 20.

Design of Cell-Permeable Inhibitors of Eukaryotic Translation Initiation Factor 4E (eIF4E) for Inhibiting Aberrant Cap-Dependent Translation in Cancer.

Emilio L Cárdenas, Rachel L O'Rourke, Arya Menon, Jennifer Meagher, Jeanne Stuckey, Amanda L Garner.

Eukaryotic translation initiation factor 4E (eIF4E) is an RNA-binding protein that binds to the m7GpppX-cap at the 5' terminus of coding mRNAs to initiate cap-dependent translation. While all cells require cap-dependent translation, cancer cells become addicted to enhanced translational capacity, driving the production of oncogenic proteins involved in proliferation, evasion of apoptosis, metastasis, and angiogenesis, among other cancerous phenotypes. eIF4E is the rate-limiting translation factor, and its activation has been shown to drive cancer initiation, progression, metastasis, and drug resistance. These findings have established eIF4E as a translational oncogene and promising, albeit challenging, anti-cancer therapeutic target. Although significant effort has been put forth toward inhibiting eIF4E, the design of cell-permeable, cap-competitive inhibitors remains a challenge. Herein, we describe our work toward solving this long-standing challenge. By employing an acyclic nucleoside phosphonate prodrug strategy, we report the synthesis of cell-permeable inhibitors of eIF4E binding to capped mRNA to inhibit cap-dependent translation.

DOI: https://doi.org/10.1021/acs.jmedchem.3c00917
Nature. 2023 Jul 19.

Mega-scale experimental analysis of protein folding stability in biology and design.

Kotaro Tsuboyama, Justas Dauparas, Jonathan Chen, Elodie Laine, Yasser Mohseni Behbahani, Jonathan J Weinstein, Niall M Mangan, Sergey Ovchinnikov, Gabriel J Rocklin.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

DOI: https://doi.org/10.1038/s41586-023-06328-6
EMBO Rep. 2023 Jul 19. e57289

Autophagy orchestrates the crosstalk between cells and organs.

Klara Piletic, Ghada Alsaleh, Anna Katharina Simon.

  Over the recent years, it has become apparent that a deeper understanding of cell-to-cell and organ-to-organ communication is necessary to fully comprehend both homeostatic and pathological states. Autophagy is indispensable for cellular development, function, and homeostasis. A crucial aspect is that autophagy can also mediate these processes through its secretory role. The autophagy-derived secretome relays its extracellular signals in the form of nutrients, proteins, mitochondria, and extracellular vesicles. These crosstalk mediators functionally shape cell fate decisions, tissue microenvironment and systemic physiology. The diversity of the secreted cargo elicits an equally diverse type of responses, which span over metabolic, inflammatory, and structural adaptations in disease and homeostasis. We review here the emerging role of the autophagy-derived secretome in the communication between different cell types and organs and discuss the mechanisms involved.

Keywords:  EV secretion; autophagy-derived secretome; intercellular communication; secretory autophagy; unconventional secretion

DOI:  https://doi.org/10.15252/embr.202357289
RNA. 2023 Jul 17. pii: rna.079755.123. [Epub ahead of print]

Phase separated ribosome-nascent chain complexes in genotoxic stress response.

Orsolya Nemeth-Szatmari, Bence Nagy-Miko, Adam Gyorkei, Daniel Varga, Balint Barna H Kovacs, Nora Igaz, Bence Bognar, Zsolt Razga, Gabor Nagy, Nora Zsindely, Laszlo Bodai, Balazs Papp, Miklos Erdelyi, Monika Kiricsi, Andras Blastyak, Martine A Collart, Imre Miklos Boros, Zoltan Villanyi.

  Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered N-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their N-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

Keywords:  BLM; DNA damage response; Sgs1; assemblysomes; phase-separation

DOI:  https://doi.org/10.1261/rna.079755.123