bims-proteo 2023-07-02 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–07–02
25 papers selected by
Eric Chevet, INSERM

A novel endoplasmic reticulum adaptation is critical for the long-lived Caenorhabditis elegans rpn-10 proteasomal mutant.
The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins.
ULK phosphorylation of STX17 controls autophagosome maturation via FLNA.
Interactome Analysis of the ER Stress Sensor Perk Uncovers Key Components of ER-Mitochondria Contact Sites and Ca2+ Signalling.
Beyond UPR: Cell-specific Roles of ER Stress Sensor IRE1α in Kidney Ischemic Injury and Transplant Rejection.
Dynamic mapping of proteome trafficking within and between living cells by TransitID.
Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization.
An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes.
Molecular Highway Patrol for Ribosome Collisions.
ECPAS/Ecm29-mediated 26S proteasome disassembly is an adaptive response to glucose starvation.
Oligomeric Remodeling by Molecular Glues Revealed Using Native Mass Spectrometry and Mass Photometry.
Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.
A membrane-sensing mechanism links lipid metabolism to protein degradation at the nuclear envelope.
Uncovering the Kinetic Characteristics and Degradation Preference of PROTAC Systems with Advanced Theoretical Analyses.
Circadian clocks are modulated by compartmentalized oscillating translation.
Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification.
Proteins rather than mRNAs regulate nucleation and persistence of Oskar germ granules in Drosophila.
Cell death or survival: Insights into the role of mRNA translational control.
Autism-linked UBE3A gain-of-function mutation causes interneuron and behavioral phenotypes when inherited maternally or paternally in mice.
Gbb glutathionylation promotes its proteasome-mediated degradation to inhibit synapse growth.
Protein kinase ATR inhibits E3 ubiquitin ligase CRL4PRL1 to stabilize ribonucleotide reductase in response to replication stress.
TSignal: a transformer model for signal peptide prediction.
Coupled deglycosylation-ubiquitination cascade in regulating PD-1 degradation by MDM2.
An engineered cell line with a hRpn1-attached handle to isolate proteasomes.
Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel.

Biochim Biophys Acta Gene Regul Mech. 2023 Jun 22. pii: S1874-9399(23)00052-4. [Epub ahead of print] 194957

A novel endoplasmic reticulum adaptation is critical for the long-lived Caenorhabditis elegans rpn-10 proteasomal mutant.

Meghna N Chinchankar, William B Taylor, Su-Hyuk Ko, Ellen C Apple, Karl A Rodriguez, Lizhen Chen, Alfred L Fisher.

  The loss of proteostasis due to reduced efficiency of protein degradation pathways plays a key role in multiple age-related diseases and is a hallmark of the aging process. Paradoxically, we have reported that the Caenorhabditis elegans rpn-10(ok1865) mutant, which lacks the RPN-10/RPN10/PSMD4 subunit of the 19S regulatory particle of the 26S proteasome, exhibits enhanced cytosolic proteostasis, elevated stress resistance and extended lifespan, despite possessing reduced proteasome function. However, the response of this mutant against threats to endoplasmic reticulum (ER) homeostasis and proteostasis was unknown. Here, we find that the rpn-10 mutant is highly ER stress resistant compared to the wildtype. Under unstressed conditions, the ER unfolded protein response (UPR) is activated in the rpn-10 mutant as signified by increased xbp-1 splicing. This primed response appears to alter ER homeostasis through the upregulated expression of genes involved in ER protein quality control (ERQC), including those in the ER-associated protein degradation (ERAD) pathway. Pertinently, we find that ERQC is critical for the rpn-10 mutant longevity. These changes also alter ER proteostasis, as studied using the C. elegans alpha-1 antitrypsin (AAT) deficiency model, which comprises an intestinal ER-localised transgenic reporter of an aggregation-prone form of AAT called ATZ. The rpn-10 mutant shows a significant reduction in the accumulation of the ATZ reporter, thus indicating that its ER proteostasis is augmented. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we then identified ecps-2/H04D03.3, a novel ortholog of the proteasome-associated adaptor and scaffold protein ECM29/ECPAS. We further show that ecps-2 is required for improved ER proteostasis as well as lifespan extension of the rpn-10 mutant. Thus, we propose that ECPS-2-proteasome functional interactions, alongside additional putative molecular processes, contribute to a novel ERQC adaptation which underlies the superior proteostasis and longevity of the rpn-10 mutant.

Keywords:  Caenorhabditis elegans; ECPS-2/ECM29/ECPAS; Endoplasmic reticulum (ER); Proteasome; RPN-10/RPN10/PSMD4

DOI:  https://doi.org/10.1016/j.bbagrm.2023.194957
Mol Biol Cell. 2023 Jun 28. mbcE23050205

The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins.

Katharina Knöringer, Carina Groh, Lena Krämer, Kevin C Stein, Katja G Hansen, Jannik Zimmermann, Bruce Morgan, Johannes M Herrmann, Judith Frydman, Felix Boos.

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPRER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The level of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPRER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cannot be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

DOI: https://doi.org/10.1091/mbc.E23-05-0205
J Cell Biol. 2023 Aug 07. pii: e202211025. [Epub ahead of print]222(8):

ULK phosphorylation of STX17 controls autophagosome maturation via FLNA.

Yufen Wang, Huilin Que, ChuangPeng Li, Zhe Wu, Fenglei Jian, Yuan Zhao, Haohao Tang, Yang Chen, Shuaixin Gao, Catherine C L Wong, Ying Li, Chongchong Zhao, Yueguang Rong.

Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in the late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitating autophagosome-lysosome fusion. Disease-causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.

DOI: https://doi.org/10.1083/jcb.202211025
Contact (Thousand Oaks). 2021 Jan-Dec;4:4 25152564211052392

Interactome Analysis of the ER Stress Sensor Perk Uncovers Key Components of ER-Mitochondria Contact Sites and Ca2+ Signalling.

Maria Livia Sassano, Rita Derua, Etienne Waelkens, Patrizia Agostinis, Alexander R van Vliet.

  We recently reported that the ER stress kinase PERK regulates ER-mitochondria appositions and ER- plasma membrane (ER-PM) contact sites, independent of its canonical role in the unfolded protein response. PERK regulation of ER-PM contacts was revealed by a proximity biotinylation (BioID) approach and involved a dynamic PERK-Filamin A interaction supporting the formation of ER-PM contacts by actin-cytoskeleton remodeling in response to depletion of ER-Ca2+ stores. In this report, we further interrogated the PERK BioID interactome by validating through co-IP experiments the interaction between PERK and two proteins involved in Ca2+ handling and ER-mitochondria contact sites. These included the vesicle associated membrane (VAMP)-associated proteins (VAPA/B) and the main ER Ca2+ pump sarcoplasmic/endoplasmic reticulum Ca ATPase 2 (SERCA2). These data identify new putative PERK interacting proteins with a crucial role in membrane contact sites and Ca2+ signaling further supporting the uncanonical role of PERK in Ca2+ signaling through membrane contact sites (MCSs).

Keywords:  ER stress; cell biology; endoplasmic reticulum; mitochondrial associated membranes (MAM); sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)

DOI:  https://doi.org/10.1177/25152564211052392
Kidney Int. 2023 Jun 28. pii: S0085-2538(23)00470-2. [Epub ahead of print]

Beyond UPR: Cell-specific Roles of ER Stress Sensor IRE1α in Kidney Ischemic Injury and Transplant Rejection.

Longhui Qiu, Xin Zheng, Dinesh Jaishankar, Richard Green, Deyu Fang, Satish Nadig, Zheng Jenny Zhang.

  Kidney damage due to ischemia or rejection results in the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) lumen, a condition known as "ER stress". Inositol-requiring enzyme 1α (IRE1α), the first ER stress sensor found, is a type I transmembrane protein with kinase and endoribonuclease activity. Upon activation, IRE1α non-conventionally splices an intron from unspliced X-box binding protein 1 (XBP1u) mRNA to produce XBP1s mRNA that encodes the transcription factor, XBP1s, for the expression of genes encoding proteins that mediate the unfolded protein response (UPR). The UPR promotes the functional fidelity of ER and is required for secretory cells to sustain protein folding and secretory capability. Prolonged ER stress can lead to apoptosis, which may result in detrimental repercussions to organ health and has been implicated in the pathogenesis and progression of kidney diseases. The IRE1α-XBP1 signaling acts as a major arm of UPR and is involved in regulating autophagy, cell differentiation, and cell death. IRE1α also interacts with Activator Protein-1 (AP-1) and Nuclear Factor-κB (NF-κB) pathways to regulate inflammatory responses. Studies using transgenic mouse models highlight that the roles of IRE1α differ depending on cell type and disease setting. This review covers these cell-specific roles of IRE1α signaling and the potential for therapeutic targeting of this pathway in the context of ischemia and rejection affecting the kidneys.

Keywords:  Acute kidney injury; Cell signaling; Cell survival; Endoplasmic reticulum; Transplantation

DOI:  https://doi.org/10.1016/j.kint.2023.06.016
Cell. 2023 Jun 23. pii: S0092-8674(23)00596-2. [Epub ahead of print]

Dynamic mapping of proteome trafficking within and between living cells by TransitID.

Wei Qin, Joleen S Cheah, Charles Xu, James Messing, Brian D Freibaum, Steven Boeynaems, J Paul Taylor, Namrata D Udeshi, Steven A Carr, Alice Y Ting.

  The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

Keywords:  JUN; intercellular signaling; membraneless organelles; protein trafficking; proximity labeling; spatial proteomics; stress granules; tumor-associated macrophages

DOI:  https://doi.org/10.1016/j.cell.2023.05.044
Cell Chem Biol. 2023 Jun 20. pii: S2451-9456(23)00157-5. [Epub ahead of print]

Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization.

Martin P Schwalm, Andreas Krämer, Anja Dölle, Janik Weckesser, Xufen Yu, Jian Jin, Krishna Saxena, Stefan Knapp.

  The multi-step degradation process of PROteolysis TArgeting Chimeras (PROTACs) poses a challenge for their rational development, as the rate-limiting steps that determine PROTACs efficiency remain largely unknown. Moreover, the slow throughput of currently used endpoint assays does not allow the comprehensive analysis of larger series of PROTACs. Here, we developed cell-based assays using the NanoLuciferase and HaloTag that allow measuring PROTAC-induced degradation and ternary complex formation kinetics and stability in cells. Using PROTACs developed for the degradation of WD40 repeat domain protein 5 (WDR5), the characterization of the mode of action of these PROTACs in the early degradation cascade revealed a key role of ternary complex formation and stability. Comparing a series of ternary complex crystal structures highlighted the importance of an efficient E3-target interface for ternary complex stability. The developed assays outline a strategy for the rational optimization of PROTACs using a series of live cell assays monitoring key steps of the early PROTAC-induced degradation pathway.

Keywords:  HiBiT; NanoBiT; PROTAC; Targeted Protein Degradation; WDR5; degradation kinetics; degrader; kinetics; live cells; screening

DOI:  https://doi.org/10.1016/j.chembiol.2023.06.002
EMBO Rep. 2023 Jun 29. e56841

An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes.

Dale P Corkery, Sergio Castro-Gonzalez, Anastasia Knyazeva, Laura K Herzog, Yao-Wen Wu.

  Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin-dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome-lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N-terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3-like conjugation complex with the ATG12-ATG5 conjugate to regulate ATG16L1-independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.

Keywords:  TECPR1; autophagy; lysophagy; lysosome; membrane repair

DOI:  https://doi.org/10.15252/embr.202356841
Chembiochem. 2023 Jun 29. e202300264

Molecular Highway Patrol for Ribosome Collisions.

Kaushik Viswanathan Iyer, Max Müller, Lena Sophie Tittel, Marie-Luise Winz.

  During translation, messenger RNAs (mRNAs) are decoded by ribosomes which can stall for various reasons. These include chemical damage, codon composition, starvation, or translation inhibition. Trailing ribosomes can collide with stalled ribosomes, potentially leading to dysfunctional or toxic proteins. Such aberrant proteins can form aggregates and favor diseases, especially neurodegeneration. To prevent this, both eukaryotes and bacteria have evolved different pathways to remove faulty nascent peptides, mRNAs and defective ribosomes from the collided complex. In eukaryotes, ubiquitin ligases play central roles in triggering downstream responses and several complexes have been characterized that split affected ribosomes and facilitate degradation of the various components. As collided ribosomes signal translation stress to affected cells, in eukaryotes additional stress response pathways are triggered when collisions are sensed. These pathways inhibit translation and modulate cell survival and immune responses. Here, we summarize the current state of knowledge about rescue and stress response pathways triggered by ribosome collisions.

Keywords:  RNA; RQC; protein; stress response; translation quality control

DOI:  https://doi.org/10.1002/cbic.202300264
Cell Rep. 2023 Jun 27. pii: S2211-1247(23)00712-X. [Epub ahead of print]42(7): 112701

ECPAS/Ecm29-mediated 26S proteasome disassembly is an adaptive response to glucose starvation.

Won Hoon Choi, Yejin Yun, Insuk Byun, Sumin Kim, Seho Lee, Jiho Sim, Shahar Levi, Seo Hyeong Park, Jeongmoo Jun, Oded Kleifeld, Kwang Pyo Kim, Dohyun Han, Tomoki Chiba, Chaok Seok, Yong Tae Kwon, Michael H Glickman, Min Jae Lee.

  The 26S proteasome comprises 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in cells exist as free 20S complexes; however, our mechanistic understanding of what determines the ratio of 26S to 20S species remains incomplete. Here, we show that glucose starvation uncouples 26S holoenzymes into 20S and 19S subcomplexes. Subcomplex affinity purification and quantitative mass spectrometry reveal that Ecm29 proteasome adaptor and scaffold (ECPAS) mediates this structural remodeling. The loss of ECPAS abrogates 26S dissociation, reducing degradation of 20S proteasome substrates, including puromycylated polypeptides. In silico modeling suggests that ECPAS conformational changes commence the disassembly process. ECPAS is also essential for endoplasmic reticulum stress response and cell survival during glucose starvation. In vivo xenograft model analysis reveals elevated 20S proteasome levels in glucose-deprived tumors. Our results demonstrate that the 20S-19S disassembly is a mechanism adapting global proteolysis to physiological needs and countering proteotoxic stress.

Keywords:  19S complex; 20S proteasome; CP: Metabolism; ECPAS; Ecm29; disassembly; glucose; in silico modeling; proteasome; proteolysis; starvation

DOI:  https://doi.org/10.1016/j.celrep.2023.112701
J Am Chem Soc. 2023 Jun 28.

Oligomeric Remodeling by Molecular Glues Revealed Using Native Mass Spectrometry and Mass Photometry.

Xiaojing Huang, Hari Kamadurai, Piro Siuti, Ezaz Ahmed, Jack L Bennett, William A Donald.

Molecular glues stabilize interactions between E3 ligases and novel substrates to promote substrate degradation, thereby facilitating the inhibition of traditionally "undruggable" protein targets. However, most known molecular glues have been discovered fortuitously or are based on well-established chemical scaffolds. Efficient approaches for discovering and characterizing the effects of molecular glues on protein interactions are required to accelerate the discovery of novel agents. Here, we demonstrate that native mass spectrometry and mass photometry can provide unique insights into the physical mechanism of molecular glues, revealing previously unknown effects of such small molecules on the oligomeric organization of E3 ligases. When compared to well-established solution phase assays, native mass spectrometry provides accurate quantitative descriptions of molecular glue potency and efficacy while also enabling the binding specificity of E3 ligases to be determined in a single, rapid measurement. Such mechanistic insights should accelerate the rational development of molecular glues to afford powerful therapeutic agents.

DOI: https://doi.org/10.1021/jacs.3c02655
Mol Cell Proteomics. 2023 Jun 27. pii: S1535-9476(23)00120-2. [Epub ahead of print] 100609

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.

Darragh P O'Brien, Hannah Bl Jones, Franziska Guenther, Emma J Murphy, Katherine S England, Iolanda Vendrell, Malcolm Anderson, Paul Brennan, John B Davis, Adán Pinto-Fernández, Andrew P Turnbull, Benedikt M Kessler.

Dampening functional levels of the mitochondrial deubiquitylating enzyme USP30 has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide containing compound, USP30inh. Activity-based protein profiling (ABPP) mass spectrometry confirmed target engagement, the high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics infers slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to the thumb-palm cleft that guides the ubiquitin C-terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

DOI: https://doi.org/10.1016/j.mcpro.2023.100609
J Cell Biol. 2023 Sep 04. pii: e202304026. [Epub ahead of print]222(9):

A membrane-sensing mechanism links lipid metabolism to protein degradation at the nuclear envelope.

Shoken Lee, Jake W Carrasquillo Rodrı Guez, Holly Merta, Shirin Bahmanyar.

Lipid composition determines organelle identity; however, whether the lipid composition of the inner nuclear membrane (INM) domain of the ER contributes to its identity is not known. Here, we show that the INM lipid environment of animal cells is under local control by CTDNEP1, the master regulator of the phosphatidic acid phosphatase lipin 1. Loss of CTDNEP1 reduces association of an INM-specific diacylglycerol (DAG) biosensor and results in a decreased percentage of polyunsaturated containing DAG species. Alterations in DAG metabolism impact the levels of the resident INM protein Sun2, which is under local proteasomal regulation. We identify a lipid-binding amphipathic helix (AH) in the nucleoplasmic domain of Sun2 that prefers membrane packing defects. INM dissociation of the Sun2 AH is linked to its proteasomal degradation. We suggest that direct lipid-protein interactions contribute to sculpting the INM proteome and that INM identity is adaptable to lipid metabolism, which has broad implications on disease mechanisms associated with the nuclear envelope.

DOI: https://doi.org/10.1083/jcb.202304026
JACS Au. 2023 Jun 26. 3(6): 1775-1789

Uncovering the Kinetic Characteristics and Degradation Preference of PROTAC Systems with Advanced Theoretical Analyses.

Rongfan Tang, Zhe Wang, Sutong Xiang, Lingling Wang, Yang Yu, Qinghua Wang, Qirui Deng, Tingjun Hou, Huiyong Sun.

Proteolysis-targeting chimeras (PROTACs), which can selectively induce the degradation of target proteins, represent an attractive technology in drug discovery. A large number of PROTACs have been reported, but due to the complicated structural and kinetic characteristics of the target-PROTAC-E3 ligase ternary interaction process, the rational design of PROTACs is still quite challenging. Here, we characterized and analyzed the kinetic mechanism of MZ1, a PROTAC that targets the bromodomain (BD) of the bromodomain and extra terminal (BET) protein (Brd2, Brd3, or Brd4) and von Hippel-Lindau E3 ligase (VHL), from the kinetic and thermodynamic perspectives of view by using enhanced sampling simulations and free energy calculations. The simulations yielded satisfactory predictions on the relative residence time and standard binding free energy (rp > 0.9) for MZ1 in different BrdBD-MZ1-VHL ternary complexes. Interestingly, the simulation of the PROTAC ternary complex disintegration illustrates that MZ1 tends to remain on the surface of VHL with the BD proteins dissociating alone without a specific dissociation direction, indicating that the PROTAC prefers more to bind with E3 ligase at the first step in the formation of the target-PROTAC-E3 ligase ternary complex. Further exploration of the degradation difference of MZ1 in different Brd systems shows that the PROTAC with higher degradation efficiency tends to leave more lysine exposed on the target protein, which is guaranteed by the stability (binding affinity) and durability (residence time) of the target-PROTAC-E3 ligase ternary complex. It is quite possible that the underlying binding characteristics of the BrdBD-MZ1-VHL systems revealed by this study may be shared by different PROTAC systems as a general rule, which may accelerate rational PROTAC design with higher degradation efficiency.

DOI: https://doi.org/10.1021/jacsau.3c00195
Cell. 2023 Jun 20. pii: S0092-8674(23)00599-8. [Epub ahead of print]

Circadian clocks are modulated by compartmentalized oscillating translation.

Yanrong Zhuang, Zhiyuan Li, Shiyue Xiong, Chujie Sun, Boya Li, Shuangcheng Alivia Wu, Jiali Lyu, Xiang Shi, Liang Yang, Yutong Chen, Zhangbin Bao, Xi Li, Chuhanwen Sun, Yuling Chen, Haiteng Deng, Tingting Li, Qingfeng Wu, Ling Qi, Yue Huang, Xuerui Yang, Yi Lin.

  Terrestrial organisms developed circadian rhythms for adaptation to Earth's quasi-24-h rotation. Achieving precise rhythms requires diurnal oscillation of fundamental biological processes, such as rhythmic shifts in the cellular translational landscape; however, regulatory mechanisms underlying rhythmic translation remain elusive. Here, we identified mammalian ATXN2 and ATXN2L as cooperating master regulators of rhythmic translation, through oscillating phase separation in the suprachiasmatic nucleus along circadian cycles. The spatiotemporal oscillating condensates facilitate sequential initiation of multiple cycling processes, from mRNA processing to protein translation, for selective genes including core clock genes. Depleting ATXN2 or 2L induces opposite alterations to the circadian period, whereas the absence of both disrupts translational activation cycles and weakens circadian rhythmicity in mice. Such cellular defect can be rescued by wild type, but not phase-separation-defective ATXN2. Together, we revealed that oscillating translation is regulated by spatiotemporal condensation of two master regulators to achieve precise circadian rhythm in mammals.

Keywords:  ATXN2; ATXN2L; circadian rhythm; clock genes; membrane-less organelle; neurodegeneration; phase separation; rhythmic translation; suprachiasmatic nucleus; translational regulation

DOI:  https://doi.org/10.1016/j.cell.2023.05.045
Nucleic Acids Res. 2023 Jun 28. pii: gkad545. [Epub ahead of print]

Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification.

Hirotatsu Imai, Daisuke Utsumi, Hidetsugu Torihara, Kenzo Takahashi, Hidehito Kuroyanagi, Akio Yamashita.

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires nascent RNA synthesis and translation. Analysis of the coordinated regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprogramming of nascent RNA synthesis and translation. Our nascent P-TRAP (nP-TRAP) method may serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.

DOI: https://doi.org/10.1093/nar/gkad545
Cell Rep. 2023 Jun 28. pii: S2211-1247(23)00734-9. [Epub ahead of print]42(7): 112723

Proteins rather than mRNAs regulate nucleation and persistence of Oskar germ granules in Drosophila.

Harrison A Curnutte, Xinyue Lan, Manuel Sargen, Si Man Ao Ieong, Dylan Campbell, Hyosik Kim, Yijun Liao, Sarah Bailah Lazar, Tatjana Trcek.

  RNA granules are membraneless condensates that provide functional compartmentalization within cells. The mechanisms by which RNA granules form are under intense investigation. Here, we characterize the role of mRNAs and proteins in the formation of germ granules in Drosophila. Super-resolution microscopy reveals that the number, size, and distribution of germ granules is precisely controlled. Surprisingly, germ granule mRNAs are not required for the nucleation or the persistence of germ granules but instead control their size and composition. Using an RNAi screen, we determine that RNA regulators, helicases, and mitochondrial proteins regulate germ granule number and size, while the proteins of the endoplasmic reticulum, nuclear pore complex, and cytoskeleton control their distribution. Therefore, the protein-driven formation of Drosophila germ granules is mechanistically distinct from the RNA-dependent condensation observed for other RNA granules such as stress granules and P-bodies.

Keywords:  CP: Cell biology; ER; Oskar; RNA condensates; RNA granules; Vasa; condensates; germ granules; nanos; nuclear pores; phase separation; polar granules

DOI:  https://doi.org/10.1016/j.celrep.2023.112723
Semin Cell Dev Biol. 2023 Jun 23. pii: S1084-9521(23)00133-7. [Epub ahead of print]

Cell death or survival: Insights into the role of mRNA translational control.

Nupur Bhatter, Sergey E Dmitriev, Pavel Ivanov.

  Cellular stress is an intrinsic part of cell physiology that underlines cell survival or death. The ability of mammalian cells to regulate global protein synthesis (aka translational control) represents a critical, yet underappreciated, layer of regulation during the stress response. Various cellular stress response pathways monitor conditions of cell growth and subsequently reshape the cellular translatome to optimize translational outputs. On the molecular level, such translational reprogramming involves an intricate network of interactions between translation machinery, RNA-binding proteins, mRNAs, and non-protein coding RNAs. In this review, we will discuss molecular mechanisms, signaling pathways, and targets of translational control that contribute to cellular adaptation to stress and to cell survival or death.

Keywords:  RNA modifications; Stress granules; eIF2α phosphorylation; mRNA translation; mTOR; uORFs

DOI:  https://doi.org/10.1016/j.semcdb.2023.06.006
Cell Rep. 2023 Jun 28. pii: S2211-1247(23)00717-9. [Epub ahead of print]42(7): 112706

Autism-linked UBE3A gain-of-function mutation causes interneuron and behavioral phenotypes when inherited maternally or paternally in mice.

Lei Xing, Jeremy M Simon, Travis S Ptacek, Jason J Yi, Lipin Loo, Hanqian Mao, Justin M Wolter, Eric S McCoy, Smita R Paranjape, Bonnie Taylor-Blake, Mark J Zylka.

  The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.

Keywords:  Angelman syndrome; CP: Neuroscience; UBE3A; autism; neurodevelopmental disorders; single-cell RNA-seq

DOI:  https://doi.org/10.1016/j.celrep.2023.112706
J Cell Biol. 2023 Sep 04. pii: e202202068. [Epub ahead of print]222(9):

Gbb glutathionylation promotes its proteasome-mediated degradation to inhibit synapse growth.

Md Shafayat Hossain, Aiyu Yao, Xinhua Qiao, Wenwen Shi, Ting Xie, Chang Chen, Yong Q Zhang.

Glutathionylation is a posttranslational modification involved in various molecular and cellular processes. However, it remains unknown whether and how glutathionylation regulates nervous system development. To identify critical regulators of synapse growth and development, we performed an RNAi screen and found that postsynaptic knockdown of glutathione transferase omega 1 (GstO1) caused significantly more synaptic boutons at the Drosophila neuromuscular junctions. Genetic and biochemical analysis revealed an increased level of glass boat bottom (Gbb), the Drosophila homolog of mammalian bone morphogenetic protein (BMP), in GstO1 mutants. Further experiments showed that GstO1 is a critical regulator of Gbb glutathionylation at cysteines 354 and 420, which promoted its degradation via the proteasome pathway. Moreover, the E3 ligase Ctrip negatively regulated the Gbb protein level by preferentially binding to glutathionylated Gbb. These results unveil a novel regulatory mechanism in which glutathionylation of Gbb facilitates its ubiquitin-mediated degradation. Taken together, our findings shed new light on the crosstalk between glutathionylation and ubiquitination of Gbb in synapse development.

DOI: https://doi.org/10.1083/jcb.202202068
Cell Rep. 2023 Jun 23. pii: S2211-1247(23)00696-4. [Epub ahead of print]42(7): 112685

Protein kinase ATR inhibits E3 ubiquitin ligase CRL4PRL1 to stabilize ribonucleotide reductase in response to replication stress.

Weiyi Bao, Weijia Zhang, Yongchi Huang, Yan Zhao, Cong Wu, Leilei Duan, Lili Wang, Shunping Yan.

  The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide (dNTP), the universal building block for DNA replication and repair. However, the relationship between ATR and RNR is not well understood. Here, we show that ATR promotes the protein stability of RNR in Arabidopsis. Through an activation tagging-based genetic screen, we found that overexpression of TSO2, a small subunit of RNR, partially suppresses the hypersensitivity of the atr mutant to replication stress. Biochemically, TSO2 interacts with PRL1, a central subunit of the Cullin4-based E3 ubiquitin ligase CRL4PRL1, which polyubiquitinates TSO2 and promotes its degradation. ATR inhibits CRL4PRL1 to attenuate TSO2 degradation. Our work provides an important insight into the replication stress responses and a post-translational regulatory mechanism for RNR. Given the evolutionary conservation of the proteins involved, the ATR-PRL1-RNR module may act across eukaryotes.

Keywords:  ATR; CP: Molecular biology; DNA damage response; DNA replication stress; PRL1; TSO2; WEE1; ribonucleotide reductase

DOI:  https://doi.org/10.1016/j.celrep.2023.112685
Bioinformatics. 2023 Jun 30. 39(Supplement_1): i347-i356

TSignal: a transformer model for signal peptide prediction.

Alexandru Dumitrescu, Emmi Jokinen, Anja Paatero, Juho Kellosalo, Ville O Paavilainen, Harri Lähdesmäki.

MOTIVATION: Signal peptides (SPs) are short amino acid segments present at the N-terminus of newly synthesized proteins that facilitate protein translocation into the lumen of the endoplasmic reticulum, after which they are cleaved off. Specific regions of SPs influence the efficiency of protein translocation, and small changes in their primary structure can abolish protein secretion altogether. The lack of conserved motifs across SPs, sensitivity to mutations, and variability in the length of the peptides make SP prediction a challenging task that has been extensively pursued over the years.
RESULTS: We introduce TSignal, a deep transformer-based neural network architecture that utilizes BERT language models and dot-product attention techniques. TSignal predicts the presence of SPs and the cleavage site between the SP and the translocated mature protein. We use common benchmark datasets and show competitive accuracy in terms of SP presence prediction and state-of-the-art accuracy in terms of cleavage site prediction for most of the SP types and organism groups. We further illustrate that our fully data-driven trained model identifies useful biological information on heterogeneous test sequences.
AVAILABILITY AND IMPLEMENTATION: TSignal is available at: https://github.com/Dumitrescu-Alexandru/TSignal.

DOI: https://doi.org/10.1093/bioinformatics/btad228
Cell Rep. 2023 Jun 27. pii: S2211-1247(23)00704-0. [Epub ahead of print]42(7): 112693

Coupled deglycosylation-ubiquitination cascade in regulating PD-1 degradation by MDM2.

Zhen Wu, Zhijie Cao, Han Yao, Xiaojun Yan, Wenbin Xu, Mi Zhang, Zishan Jiao, Zijing Zhang, Jianyuan Chen, Yajing Liu, Meng Zhang, Donglai Wang.

  Posttranslational modifications represent a key step in modulating programmed death-1 (PD-1) functions, but the underlying mechanisms remain incompletely defined. Here, we report crosstalk between deglycosylation and ubiquitination in regulating PD-1 stability. We show that the removal of N-linked glycosylation is a prerequisite for efficient PD-1 ubiquitination and degradation. Murine double minute 2 (MDM2) is identified as an E3 ligase of deglycosylated PD-1. In addition, the presence of MDM2 facilitates glycosylated PD-1 interaction with glycosidase NGLY1 and promotes subsequent NGLY1-catalyzed PD-1 deglycosylation. Functionally, we demonstrate that the absence of T cell-specific MDM2 accelerates tumor growth by primarily upregulating PD-1. By stimulating the p53-MDM2 axis, interferon-α (IFN-α) reduces PD-1 levels in T cells, which, in turn, exhibit a synergistic effect on tumor suppression by sensitizing anti-PD-1 immunotherapy. Our study reveals that MDM2 directs PD-1 degradation via a deglycosylation-ubiquitination coupled mechanism and sheds light on a promising strategy to boost cancer immunotherapy by targeting the T cell-specific MDM2-PD-1 regulatory axis.

Keywords:  CP: Cancer; CP: Molecular biology; MDM2; NGLY1; PD-1; antitumor immunity; deglycosylation; ubiquitination

DOI:  https://doi.org/10.1016/j.celrep.2023.112693
J Biol Chem. 2023 Jun 22. pii: S0021-9258(23)01976-2. [Epub ahead of print] 104948

An engineered cell line with a hRpn1-attached handle to isolate proteasomes.

Hitendra Negi, Vasty Osei-Amponsa, Bishoy Ibrahim, Christine N Evans, Catherine Sullenberger, Jadranka Loncarek, Raj Chari, Kylie J Walters.

  Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which contains a 28-subunit hollow core particle (CP) within which proteolysis occurs, and a 19-subunit regulatory particle (RP) that binds, processes, and translocates substrates to the CP. In addition to its intrinsic subunits, myriad proteins interact with the proteasome transiently, including factors that assist and/or regulate its degradative activities. Efforts to identify proteasome-interacting components and/or to solve its structure have relied on over-expression of a tagged plasmid, establishing stable cell lines, or laborious purification protocols to isolate native proteasomes from cells. Here, we describe an engineered human cell line, derived from colon cancer HCT116 cells, with a biotin handle on the RP subunit hRpn1/PSMD2 for purification of 26S proteasomes. A 75-residue sequence from Propionibacterium shermanii that is biotinylated in mammalian cells was added following a TEV protease cut site at the C-terminus of hRpn1. We tested and found that 26S proteasomes can be isolated from this modified HCT116 cell line by using a simple purification protocol. More specifically, biotinylated proteasomes were purified from the cell lysates by using neutravidin agarose resin and released from the resin following incubation with TEV protease. The purified proteasomes had equivalent activity in degrading a model ubiquitinated substrate, namely ubiquitinated p53, compared to commercially available bovine proteasomes that were purified by fractionation. In conclusion, advantages of this approach to obtain 26S proteasomes over others is the simple purification protocol and that all cellular proteins, including the tagged hRpn1 subunit, remain at endogenous stoichiometry.

Keywords:  CRISPR/Cas9; Proteasome; Regulatory particle; biotin; hRpn1

DOI:  https://doi.org/10.1016/j.jbc.2023.104948
Mol Cell. 2023 Jun 27. pii: S1097-2765(23)00426-4. [Epub ahead of print]

Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel.

Jiyuan Zhang, Weizhong Zeng, Yan Han, Wan-Ru Lee, Jen Liou, Youxing Jiang.

  Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.

Keywords:  LAMP-1 and LAMP-2; TMEM175; lysosomal LAMP proteins; lysosomal hydrolytic function; lysosomal pH homeostasis; lysosome acidification; risk factor for Parkinson’s disease

DOI:  https://doi.org/10.1016/j.molcel.2023.06.004