bims-proteo 2024-12-08 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–12–08
37 papers selected by
Eric Chevet, INSERM

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.
Identification of suitable target/E3 ligase pairs for PROTAC development using a rapamycin-induced proximity assay (RiPA).
Translation of zinc finger domains induces ribosome collision and Znf598-dependent mRNA decay in zebrafish.
Opposed regulation of endoplasmic reticulum retention under stress by ERp44 and PDIA6.
Quiescent cells maintain active degradation-mediated protein quality control requiring proteasome, autophagy and nucleus-vacuole junctions.
The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations.
IFRD1 is required for maintenance of bladder epithelial homeostasis.
Cryo-EM structure of histidyl-tRNA synthetase-like domain reveals activating crossed helices at the core of GCN2.
Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons.
Movement of the endoplasmic reticulum is driven by multiple classes of vesicles marked by Rab-GTPases.
Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32.
Target-conditioned diffusion generates potent TNFR superfamily antagonists and agonists.
Understanding ubiquitination in neurodevelopment by integrating insights across space and time.
Flaviviruses induce ER-specific remodelling of protein synthesis.
Elucidation of the late steps in the glycan-dependent ERAD of soluble misfolded glycoproteins.
Seipin governs phosphatidic acid homeostasis at the inner nuclear membrane.
How J-chain ensures the assembly of immunoglobulin IgM pentamers.
SUMO-targeted Ubiquitin Ligases as crucial mediators of protein homeostasis in Candida glabrata.
EMC3 is critical for CFTR function and calcium mobilization in the mouse intestinal epithelium.
G3BP1 promotes intermolecular RNA-RNA interactions during RNA condensation.
N-6-methyladenosine (m6A) promotes the nuclear retention of mRNAs with intact 5' splice site motifs.
β-hydroxybutyrate is a metabolic regulator of proteostasis in the aged and Alzheimer disease brain.
Structural dynamics of human ribosomes in situ reconstructed by exhaustive high-resolution template matching.
CRL3Keap1 E3 ligase facilitates ubiquitin-mediated degradation of oncogenic SRX to suppress colorectal cancer progression.
Intracellular polarization of RNAs and proteins in the human small intestinal epithelium.
Tracking transcription-translation coupling in real time.
Sequence complexity and monomer rigidity control the morphologies and aging dynamics of protein aggregates.
Engineered platelets as targeted protein degraders and application to breast cancer models.
The Yin and Yang of hsa-miR-1244 expression levels during activation of the UPR control cell fate.
Discovery of a molecular glue for EGFR degradation.
Synthetic GPCRs for programmable sensing and control of cell behaviour.
Protein engineering using variational free energy approximation.
ChREBP-mediated up-regulation of Them1 coordinates thermogenesis with glycolysis and lipogenesis in response to chronic stress.
Chronic gastroesophageal reflux dysregulates proteostasis in esophageal epithelial cells.
Cytoplasmic Aggregates of Splicing Factor Proline-Glutamine Rich Disrupt Nucleocytoplasmic Transport and Induce Persistent Stress Granules.
Yin Yang 1 and guanine quadruplexes protect dopaminergic neurons from cellular stress via transmissive dormancy.
Intricate ribosome composition and translational reprogramming in epithelial-mesenchymal transition.

Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2417390121

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.

Kelsey L Hickey, Alexandra Panov, Enya Miguel Whelan, Tillman Schäfer, Arda Mizrak, Ron R Kopito, Wolfgang Baumeister, Rubén Fernández-Busnadiego, J Wade Harper.

  A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Keywords:  protein aggregates; protein quality control; protein turnover; ubiquitin-proteasome system

DOI:  https://doi.org/10.1073/pnas.2417390121
Elife. 2024 Dec 06. pii: RP98450. [Epub ahead of print]13

Identification of suitable target/E3 ligase pairs for PROTAC development using a rapamycin-induced proximity assay (RiPA).

Bikash Adhikari, Katharina Schneider, Mathias Diebold, Christoph Sotriffer, Elmar Wolf.

  The development of proteolysis targeting chimeras (PROTACs), which induce the degradation of target proteins by bringing them into proximity with cellular E3 ubiquitin ligases, has revolutionized drug development. While the human genome encodes more than 600 different E3 ligases, current PROTACs use only a handful of them, drastically limiting their full potential. Furthermore, many PROTAC development campaigns fail because the selected E3 ligase candidates are unable to induce degradation of the particular target of interest. As more and more ligands for novel E3 ligases are discovered, the chemical effort to identify the best E3 ligase for a given target is exploding. Therefore, a genetic system to identify degradation-causing E3 ligases and suitable target/E3 ligase pairs is urgently needed. Here, we used the well-established dimerization of the FKBP12 protein and FRB domain by rapamycin to bring the target protein WDR5 into proximity with candidate E3 ligases. Strikingly, this rapamycin-induced proximity assay (RiPA) revealed that VHL, but not Cereblon, is able to induce WDR5 degradation - a finding previously made by PROTACs, demonstrating its predictive power. By optimizing the steric arrangement of all components and fusing the target protein with a minimal luciferase, RiPA can identify the ideal E3 for any target protein of interest in living cells, significantly reducing and focusing the chemical effort in the early stages of PROTAC development.

Keywords:  E3 ubiquitin ligase; PROTAC; RiPA; biochemistry; chemical biology; human; induced proximity; targeted protein degradation

DOI:  https://doi.org/10.7554/eLife.98450
PLoS Biol. 2024 Dec;22(12): e3002887

Translation of zinc finger domains induces ribosome collision and Znf598-dependent mRNA decay in zebrafish.

Kota Ishibashi, Yuichi Shichino, Peixun Han, Kimi Wakabayashi, Mari Mito, Toshifumi Inada, Seisuke Kimura, Shintaro Iwasaki, Yuichiro Mishima.

Quality control of translation is crucial for maintaining cellular and organismal homeostasis. Obstacles in translation elongation induce ribosome collision, which is monitored by multiple sensor mechanisms in eukaryotes. The E3 ubiquitin ligase Znf598 recognizes collided ribosomes, triggering ribosome-associated quality control (RQC) to rescue stalled ribosomes and no-go decay (NGD) to degrade stall-prone mRNAs. However, the impact of RQC and NGD on maintaining the translational homeostasis of endogenous mRNAs has remained unclear. In this study, we investigated the endogenous substrate mRNAs of NGD during the maternal-to-zygotic transition (MZT) of zebrafish development. RNA-Seq analysis of zebrafish znf598 mutant embryos revealed that Znf598 down-regulates mRNAs encoding the C2H2-type zinc finger domain (C2H2-ZF) during the MZT. Reporter assays and disome profiling indicated that ribosomes stall and collide while translating tandem C2H2-ZFs, leading to mRNA degradation by Znf598. Our results suggest that NGD maintains the quality of the translatome by mitigating the risk of ribosome collision at the abundantly present C2H2-ZF sequences in the vertebrate genome.

DOI: https://doi.org/10.1371/journal.pbio.3002887
Biochem J. 2024 Dec 02. pii: BCJ20240444. [Epub ahead of print]

Opposed regulation of endoplasmic reticulum retention under stress by ERp44 and PDIA6.

Boaz Tirosh, Olaya Yassin, Praveen Bellam, Odai Darawshi, Thomas LaFramboise, Miriam Shmuel, Shakti P Pattanayak, Brian K Law, Maria Hatzoglou.

  Conditions of endoplasmic reticulum (ER) stress reduce protein synthesis by provoking translation regulation, governed by the eIF2α kinase PERK. When PERK is inhibited during ER stress, retention of a selective subset of glycoproteins occurs, a phenomenon we termed selective endoplasmic reticulum retention (sERr). sERr clients are enriched with tyrosine kinase receptors (RTKs), which form large molecular weight disulfide bonded complexes in the ER. The protein disulfide isomerase ERp44 promotes sERr and increases the size of sERr complexes. Here we show that sERr is reversible upon washout. Pulse chase analyses show that upon recovery, only a small fraction of the sERr complexes disintegrates and contributes to the matured proteins, while most are newly synthesized. Sequential inductions of sERr and washouts demonstrate an accelerated recovery that is dependent on the unfolded protein response transducer IRE1. Since IRE1 regulates the expression level PDIA6, we analyzed its contribution to sERr. We found that PDIA6 and ERp44 constitutively interact by disulfides and have opposite effects on resumed recovery of trafficking following removal of sERr conditions. Deletion of ERp44 accelerates, while deletion of PDIA6 slows down recovery with a minimal effect on total protein synthesis. ERp44 is a primary interactor with sERr clients. When missing, PDIA6 partitions more into sERr complexes. Deletion of the tumor suppressor PTEN, which induces RTK signaling, promoted sERr formation kinetics, and accelerated the recovery, suggesting feedback between RTKs signaling and sERr. This study suggests that sERr, should develop physiologically or pathologically, is counteracted by adaptation responses that involve IRE1 and PDIA6.

Keywords:  ER stress; UPR; disulfides; trafficking

DOI:  https://doi.org/10.1042/BCJ20240444
J Biol Chem. 2024 Nov 29. pii: S0021-9258(24)02547-X. [Epub ahead of print] 108045

Quiescent cells maintain active degradation-mediated protein quality control requiring proteasome, autophagy and nucleus-vacuole junctions.

Dina Franić, Mihaela Pravica, Klara Zubčić, Shawna Miles, Antonio Bedalov, Mirta Boban.

  Many cells spend a major part of their life in quiescence, a reversible state characterized by a distinct cellular organization and metabolism. In glucose-depleted quiescent yeast cells, there is a metabolic shift from glycolysis to mitochondrial respiration, and a large fraction of proteasomes are reorganized into cytoplasmic granules containing disassembled particles. Given these changes, the operation of protein quality control (PQC) in quiescent cells, in particular the reliance on degradation-mediated PQC and the specific pathways involved, remains unclear. By examining model misfolded proteins expressed in glucose-depleted quiescent yeast cells, we found that misfolded proteins are targeted for selective degradation requiring functional 26S proteasomes. This indicates that a significant pool of proteasomes remains active in degrading quality control substrates. Misfolded proteins were degraded in a manner dependent on the E3 ubiquitin ligases Ubr1 and San1, with Ubr1 playing a dominant role. In contrast to exponentially growing cells, the efficient clearance of certain misfolded proteins additionally required intact nucleus-vacuole junctions (NVJ) and Cue5-independent selective autophagy. Our findings suggest that proteasome activity, autophagy, and NVJ-dependent degradation operate in parallel. Together the data demonstrate that quiescent cells maintain active PQC that relies primarily on selective protein degradation. The necessity of multiple degradation pathways for the removal of misfolded proteins during quiescence underscores the importance of misfolded protein clearance in this cellular state.

Keywords:  autophagy; inclusions; nucleus-vacuole junction; proteasome; protein aggregation; protein degradation; protein quality control; quiescence; spatial sequestration; ubiquitin

DOI:  https://doi.org/10.1016/j.jbc.2024.108045
J Cell Sci. 2024 Dec 02. pii: jcs.262019. [Epub ahead of print]

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations.

Heather A Baker, Jonathan P Bernardini, Veronika Csizmók, Angel Madero, Shriya Kamat, Hailey Eng, Jessica Lacoste, Faith Au Yeung, Sophie Comyn, Elizabeth Hui, Gaetano Calabrese, Brian Raught, Mikko Taipale, Thibault Mayor.

  Mutations can disrupt a protein's native function by causing misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms of misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors with one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant but not the wild-type protein. Our work indicates that specific missense mutations in cytosolic proteins can promote enhanced interactions with molecular chaperones. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits two distinct behaviours: acting to stabilize a wide array of cytosolic proteins including wild type variants, and specifically "buffer" some mutant proteins to reduce their turnover. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein.

Keywords:  Cytosolic quality control; DNAJA2; Missense proteins

DOI:  https://doi.org/10.1242/jcs.262019
iScience. 2024 Dec 20. 27(12): 111282

IFRD1 is required for maintenance of bladder epithelial homeostasis.

Bisiayo E Fashemi, Amala K Rougeau, Arnold M Salazar, Steven J Bark, Rayvanth Chappidi, Jeffrey W Brown, Charles J Cho, Jason C Mills, Indira U Mysorekar.

  The maintenance of homeostasis and rapid regeneration of the urothelium following stress are critical for bladder function. Here, we identify a key role for IFRD1 in maintaining urothelial homeostasis in a mouse model. We demonstrate that the murine bladder expresses IFRD1 at homeostasis, particularly in the urothelium, and its loss alters the global transcriptome with significant accumulation of endolysosomes and dysregulated uroplakin expression pattern. We show that IFRD1 interacts with mRNA-translation-regulating factors in human urothelial cells. Loss of Ifrd1 leads to disrupted proteostasis, enhanced endoplasmic reticulum (ER stress) with activation of the PERK arm of the unfolded protein response pathway, and increased oxidative stress. Ifrd1-deficient bladders exhibit urothelial cell apoptosis/exfoliation, enhanced basal cell proliferation, reduced differentiation into superficial cells, increased urothelial permeability, and aberrant voiding behavior. These findings highlight a crucial role for IFRD1 in urothelial homeostasis, suggesting its potential as a therapeutic target for bladder dysfunction.

Keywords:  Cell biology; Physiology; Transcriptomics

DOI:  https://doi.org/10.1016/j.isci.2024.111282
PNAS Nexus. 2024 Dec;3(12): pgae528

Cryo-EM structure of histidyl-tRNA synthetase-like domain reveals activating crossed helices at the core of GCN2.

Kristina Solorio-Kirpichyan, Xiao Fan, Dmitrij Golovenko, Andrei A Korostelev, Nieng Yan, Alexei Korennykh.

  GCN2 is a conserved receptor kinase activating the integrated stress response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a histidyl-tRNA synthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain (KD). How HRSL contributes to GCN2 signaling remains unknown. Here, we report a 3.2 Å cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus. This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and KDs, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. The remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This auto-inhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor.

Keywords:  GCN2; cryo-EM; integrated stress response; structure

DOI:  https://doi.org/10.1093/pnasnexus/pgae528
Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2419079121

Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons.

Frances V Hundley, Miguel A Gonzalez-Lozano, Lena M Gottschalk, Aslan N K Cook, Jiuchun Zhang, Joao A Paulo, J Wade Harper.

  Plasma membrane protein degradation and recycling are regulated by the endolysosomal system, wherein endocytic vesicles bud from the plasma membrane into the cytoplasm and mature into endosomes and then degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface and influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we develop a toolkit for capturing EEA1-positive endosomes (termed Endo-IP) and TMEM192-positive lysosomes (termed Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrate its utility by revealing the endolysosomal protein landscapes for stem cells and cortical-like iNeurons, and profiling endosomes in response to potassium-mediated neuronal depolarization. Through global profiling of endocytic cargo, we identify hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

Keywords:  endosome; iNeuron; lysosome; proteomics; stem cells

DOI:  https://doi.org/10.1073/pnas.2419079121
Mol Biol Cell. 2024 Dec 04. mbcE24040197

Movement of the endoplasmic reticulum is driven by multiple classes of vesicles marked by Rab-GTPases.

Allison Langley, Sarah Abeling-Wang, Erinn Wagner, John Salogiannis.

Peripheral endoplasmic reticulum (ER) tubules move along microtubules to interact with various organelles through membrane contact sites (MCS). Traditionally, ER moves by either sliding along stable microtubules via molecular motors or attaching to the plus ends of dynamic microtubules through tip attachment complexes (TAC). A recently discovered third process, hitchhiking, involves motile vesicles pulling ER tubules along microtubules. Previous research showed that ER hitchhikes on Rab5- and Rab7-marked endosomes, but it is uncertain if other Rab-vesicles can do the same. In U2OS cells, we screened Rabs for their ability to cotransport with ER tubules and found that ER hitchhikes on post-Golgi vesicles marked by Rab6 (isoforms a and b). Rab6-ER hitchhiking occurs independently of ER-endolysosome contacts and TAC-mediated ER movement. Depleting Rab6 and the motility of Rab6-vesicles reduces overall ER movement. Conversely, relocating these vesicles to the cell periphery causes peripheral ER accumulation, indicating that Rab6-vesicle motility is crucial for a subset of ER movements. Proximal post-Golgi vesicles marked by TGN46 are involved in Rab6-ER hitchhiking, while late Golgi vesicles (Rabs 8/10/11/13/14) are not essential for ER movement. Our further analysis finds that ER to Golgi vesicles marked by Rab1 are also capable of driving a subset of ER movements. Taken together, our findings suggest that ER hitchhiking on Rab-vesicles is a significant mode of ER movement. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E24-04-0197
Nat Commun. 2024 Dec 05. 15(1): 10633

Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32.

Samara Mishelle Ona Chuquimarca, Jonah Beenstock, Salima Daou, Jennifer Porat, Alexander F A Keszei, Jay Z Yin, Tobias Beschauner, Mark A Bayfield, Mohammad T Mazhab-Jafari, Frank Sicheri.

The enzyme complex KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) installs the universally conserved and essential N6-threonylcarbamoyl adenosine modification (t6A) on ANN-decoding tRNAs in eukaryotes and in archaea. KEOPS consists of Cgi121, Kae1, Pcc1, Gon7 and the atypical kinase/ATPase Bud32. Except Gon7, all KEOPS subunits are needed for tRNA modification, and in humans, mutations in all five genes underlie the lethal genetic disease Galloway Mowat Syndrome (GAMOS). Kae1 catalyzes the modification of tRNA, but the specific contributions of Bud32 and the other subunits are less clear. Here we solved cryogenic electron microscopy structures of KEOPS with and without a tRNA substrate. We uncover distinct flexibility of KEOPS-bound tRNA revealing a conformational change that may enable its modification by Kae1. We further identified a contact between a flipped-out base of the tRNA and an arginine residue in C-terminal tail of Bud32 that correlates with the conformational change in the tRNA. We also uncover contact surfaces within the KEOPS-tRNA holo-enzyme substrate complex that are required for Bud32 ATPase regulation and t6A modification activity. Our findings uncover inner workings of KEOPS including a basis for substrate specificity and why Kae1 depends on all other subunits.

DOI: https://doi.org/10.1038/s41467-024-54787-w
Science. 2024 Dec 06. 386(6726): 1154-1161

Target-conditioned diffusion generates potent TNFR superfamily antagonists and agonists.

Matthias Glögl, Aditya Krishnakumar, Robert J Ragotte, Inna Goreshnik, Brian Coventry, Asim K Bera, Alex Kang, Emily Joyce, Green Ahn, Buwei Huang, Wei Yang, Wei Chen, Mariana Garcia Sanchez, Brian Koepnick, David Baker.

Despite progress in designing protein-binding proteins, the shape matching of designs to targets is lower than in many native protein complexes, and design efforts have failed for the tumor necrosis factor receptor 1 (TNFR1) and other protein targets with relatively flat and polar surfaces. We hypothesized that free diffusion from random noise could generate shape-matched binders for challenging targets and tested this approach on TNFR1. We obtain designs with low picomolar affinity whose specificity can be completely switched to other family members using partial diffusion. Designs function as antagonists or as superagonists when presented at higher valency for OX40 and 4-1BB. The ability to design high-affinity and high-specificity antagonists and agonists for pharmacologically important targets in silico presages a coming era in protein design in which binders are made by computation rather than immunization or random screening approaches.

DOI: https://doi.org/10.1126/science.adp1779
Nat Struct Mol Biol. 2024 Dec 04.

Understanding ubiquitination in neurodevelopment by integrating insights across space and time.

Mateusz C Ambrozkiewicz, Sonja Lorenz.

Ubiquitination regulates a myriad of eukaryotic signaling cascades by modifying substrate proteins, thereby determining their functions and fates. In this perspective, we discuss current challenges in investigating the ubiquitin system in the developing brain. We foster the concept that ubiquitination pathways are spatiotemporally regulated and tightly intertwined with molecular and cellular transitions during neurogenesis and neural circuit assembly. Focusing on the neurologically highly relevant class of homologous to E6AP C-terminus (HECT) ubiquitin ligases, we propose cross-disciplinary translational approaches bridging state-of-the-art cell biology, proteomics, biochemistry, structural biology and neuroscience to dissect ubiquitination in neurodevelopment and its specific perturbations in brain diseases. We highlight that a comprehensive understanding of ubiquitin signaling in the brain may reveal new horizons in basic neuroscience and clinical applications.

DOI: https://doi.org/10.1038/s41594-024-01422-3
PLoS Pathog. 2024 Dec 02. 20(12): e1012766

Flaviviruses induce ER-specific remodelling of protein synthesis.

Ho Him Wong, Dorian Richard Kenneth Crudgington, Lewis Siu, Sumana Sanyal.

Flaviviruses orchestrate a unique remodelling of the endoplasmic reticulum (ER) to facilitate translation and processing of their polyprotein, giving rise to virus replication compartments. While the signal recognition particle (SRP)-dependent pathway is the canonical route for ER-targeting of nascent cellular membrane proteins, it is unknown whether flaviviruses rely on this mechanism. Here we show that Zika virus bypasses the SRP receptor via extensive interactions between the viral non-structural proteins and the host translational machinery. Remarkably, Zika virus appears to maintain ER-localised translation via NS3-SRP54 interaction instead, unlike other viruses such as influenza. Viral proteins engage SRP54 and the translocon, selectively enriching for factors supporting membrane expansion and lipid metabolism while excluding RNA binding and antiviral stress granule proteins. Our findings reveal a sophisticated viral strategy to rewire host protein synthesis pathways and create a replication-favourable subcellular niche, providing insights into viral adaptation.

DOI: https://doi.org/10.1371/journal.ppat.1012766
Plant J. 2024 Dec 06.

Elucidation of the late steps in the glycan-dependent ERAD of soluble misfolded glycoproteins.

Jennifer Schoberer, Ulrike Vavra, Yun-Ji Shin, Clemens Grünwald-Gruber, Richard Strasser.

  The endoplasmic reticulum (ER) utilizes ER-associated degradation (ERAD), a highly conserved eukaryotic pathway, to eliminate misfolded or unassembled proteins and maintain protein homeostasis in cells. The clearance of misfolded glycoproteins involves several distinct steps, including the recognition of a specific glycan signal, retrotranslocation to the cytosol, and subsequent degradation of the misfolded protein by the ubiquitin proteasome system. Confocal microscopy was used to track the fate of a well-characterized ERAD substrate via a self-complementing split fluorescent protein assay. The results demonstrate that a misfolded variant of the STRUBBELIG (SUB) extracellular protein domain (SUBEX-C57Y) is retrotranslocated to the cytosol when transiently expressed in Nicotiana benthamiana leaf epidermal cells. Retrotranslocation requires a protein domain with a lesion that is exposed in the lumen of the ER, N-glycan trimming by α-mannosidases, HRD1-mediated ubiquitination, and the ATPase function of CDC48. The retrotranslocated SUBEX-C57Y ERAD substrate undergoes deglycosylation, and proteasomal degradation is blocked by a catalytically inactive cytosolic peptide N-glycanase. These findings define distinct aspects of ERAD that have been elusive until now and may represent the default pathway for degrading misfolded glycoproteins in plants.

Keywords:  Arabidopsis thaliana; ERAD; Nicotiana benthamiana; endoplasmic reticulum; glycosylation; protein degradation; protein homeostasis

DOI:  https://doi.org/10.1111/tpj.17185
Nat Commun. 2024 Dec 02. 15(1): 10486

Seipin governs phosphatidic acid homeostasis at the inner nuclear membrane.

Anete Romanauska, Edvinas Stankunas, Maya Schuldiner, Alwin Köhler.

The nuclear envelope is a specialized subdomain of the endoplasmic reticulum and comprises the inner and outer nuclear membranes. Despite the crucial role of the inner nuclear membrane in genome regulation, its lipid metabolism remains poorly understood. Phosphatidic acid (PA) is essential for membrane growth as well as lipid storage. Using a genome-wide lipid biosensor screen in S. cerevisiae, we identify regulators of inner nuclear membrane PA homeostasis, including yeast Seipin, a known mediator of nuclear lipid droplet biogenesis. Here, we show that Seipin preserves nuclear envelope integrity by preventing its deformation and ectopic membrane formation. Mutations of specific regions of Seipin, some linked to human lipodystrophy, disrupt PA distribution at the inner nuclear membrane and nuclear lipid droplet formation. Investigating the Seipin co-factor Ldb16 reveals that a triacylglycerol binding site is crucial for lipid droplet formation, whereas PA regulation can be functionally separated. Our study highlights the potential of lipid biosensor screens for examining inner nuclear membrane lipid metabolism.

DOI: https://doi.org/10.1038/s41467-024-54811-z
EMBO J. 2024 Dec 04.

How J-chain ensures the assembly of immunoglobulin IgM pentamers.

Chiara Giannone, Xenia Mess, Ruiming He, Maria Rita Chelazzi, Annika Mayer, Anush Bakunts, Tuan Nguyen, Yevheniia Bushman, Andrea Orsi, Benedikt Gansen, Massimo Degano, Johannes Buchner, Roberto Sitia.

  Polymeric IgM immunoglobulins have high avidity for antigen and complement, and dominate primary antibody responses. They are produced either as assemblies of six µ2L2 subunits (i.e., hexamers), or as pentamers of two µ2L2 subunits and an additional protein termed J-chain (JC), which allows transcytosis across epithelia. The molecular mechanism of IgM assembly with the desired stoichiometry remained unknown. Here, we show in vitro and in cellula that JC outcompetes the sixth IgM subunit during assembly. Before insertion into IgM, JC exists as an ensemble of largely unstructured, protease-sensitive species with heterogeneous, non-native disulfide bonds. The J-chain interacts with the hydrophobic β-sheets selectively exposed by nascent pentamers. Completion of an amyloid-like core triggers JC folding and drives disulfide rearrangements that covalently stabilize JC-containing pentamers. In cells, the quality control factor ERp44 surveys IgM assembly and prevents the secretion of aberrant conformers. This mechanism allows the efficient production of high-avidity IgM for systemic or mucosal immunity.

Keywords:  Antibody Biogenesis; ERp44; Mucosal Immunity; Non-Native Disulfides; Protein Quality Control

DOI:  https://doi.org/10.1038/s44318-024-00317-9
PLoS Pathog. 2024 Dec 06. 20(12): e1012742

SUMO-targeted Ubiquitin Ligases as crucial mediators of protein homeostasis in Candida glabrata.

Dipika Gupta, Renu Shukla, Krishnaveni Mishra.

Candida glabrata is an opportunistic human pathogen, capable of causing severe systemic infections that are often resistant to standard antifungal treatments. To understand the importance of protein SUMOylation in the physiology and pathogenesis of C. glabrata, we earlier identified the components of SUMOylation pathway and demonstrated that the deSUMOylase CgUlp2 is essential for pathogenesis. In this work we show that the CgUlp2 is essential to maintain protein homeostasis via the SUMO-targeted ubiquitin ligase pathway. The dual loss of deSUMOylase and specific ubiquitin ligase, CgSlx8, results in heightened protein degradation, rendering the cells vulnerable to various stressors. This degradation affects crucial processes such as purine biosynthesis and compromises mitochondrial function in the mutants. Importantly, the absence of these ubiquitin ligases impedes the proliferation of C. glabrata in macrophages. These findings underscore the significance of SUMOylation and SUMO-mediated protein homeostasis as pivotal regulators of C. glabrata physiology and capacity to survive in host cells. Understanding these mechanisms could pave the way for the development of effective antifungal treatments.

DOI: https://doi.org/10.1371/journal.ppat.1012742
Am J Physiol Gastrointest Liver Physiol. 2024 Dec 06.

EMC3 is critical for CFTR function and calcium mobilization in the mouse intestinal epithelium.

Sarah Penrod, Xiaofang Tang, Changsuk Moon, Jeffrey A Whitsett, Anjaparavanda P Naren, Yunjie Huang.

  Membrane proteins, such as the Cystic Fibrosis Transmembrane-conductance Regulator (CFTR), play a crucial role in gastrointestinal functions and heath. Endoplasmic reticulum (ER) membrane protein complex (EMC), a multi-subunit insertase, mediates the incorporation of membrane segments into lipid bilayers during protein synthesis. Whether EMC regulates membrane proteins' processing and function in intestinal epithelial cells remains unclear. To investigate the role of EMC in the intestinal epithelium, we generated mice in which EMC subunit 3 (EMC3) was deleted in intestinal epithelial cells (EMC3ΔIEC). EMC3ΔIEC mice were viable but notable smaller compared to their wildtype littermates. While intestinal structure was generally maintained, EMC3ΔIEC crypts exhibited altered morphology, particularly at the base of the crypts with decreased goblet cells and paneth cells. Levels of multiple polytopic membrane proteins, including CFTR, were decreased in EMC3-deficient epithelial cells. Several calcium ATPase pumps were downregulated, and calcium mobilization was impaired in EMC3ΔIEC enteroids. CFTR-mediated organoid swelling in EMC3ΔIEC mice was impaired in response to both cAMP-dependent signaling and calcium-secretagogue stimulation. Our study demonstrated that EMC plays a critical role in maintaining intestinal epithelium homeostasis by regulating membrane protein biogenesis and intracellular calcium homeostasis. Maintaining intracellular calcium homeostasis may be a universal cellular function regulated by EMC.

Keywords:  Calcium; Cystic Fibrosis Transmembrane-conductance Regulator; Endoplasmic Reticulum Membrane protein Complex; Insertase; Intestinal Epithelium

DOI:  https://doi.org/10.1152/ajpgi.00066.2024
Mol Cell. 2024 Nov 26. pii: S1097-2765(24)00917-1. [Epub ahead of print]

G3BP1 promotes intermolecular RNA-RNA interactions during RNA condensation.

Dylan M Parker, Devin Tauber, Roy Parker.

  Ribonucleoprotein (RNP) granules are biomolecular condensates requiring RNA and proteins to assemble. Stress granules are RNP granules formed upon increases in non-translating messenger ribonucleoprotein particles (mRNPs) during stress. G3BP1 and G3BP2 proteins are proposed to assemble stress granules through multivalent crosslinking of RNPs. We demonstrate that G3BP1 also has "condensate chaperone" functions, which promote the assembly of stress granules but are dispensable following initial condensation. Following granule formation, G3BP1 is dispensable for the RNA component of granules to persist in vitro and in cells when RNA decondensers are inactivated. These results demonstrate that G3BP1 functions as an "RNA condenser," a protein that promotes intermolecular RNA-RNA interactions stabilizing RNA condensates, leading to RNP granule persistence. Moreover, the stability of RNA-only granules highlights the need for active mechanisms limiting RNP condensate stability and lifetime.

Keywords:  G3BP1; RNA condensation; RNP granules; condensate chaperone; intermolecular RNA interaction; stress granules

DOI:  https://doi.org/10.1016/j.molcel.2024.11.012
Life Sci Alliance. 2025 Feb;pii: e202403142. [Epub ahead of print]8(2):

N-6-methyladenosine (m6A) promotes the nuclear retention of mRNAs with intact 5' splice site motifs.

Eliza S Lee, Harrison W Smith, Yifan E Wang, Sean Sj Ihn, Leticia Scalize de Oliveira, Nevraj S Kejiou, Yijing L Liang, Syed Nabeel-Shah, Robert Y Jomphe, Shuye Pu, Jack F Greenblatt, Alexander F Palazzo.

In humans, misprocessed mRNAs containing intact 5' Splice Site (5'SS) motifs are nuclear retained and targeted for decay by ZFC3H1, a component of the Poly(A) Exosome Targeting complex, and U1-70K, a component of the U1 snRNP. In S. pombe, the ZFC3H1 homolog, Red1, binds to the YTH domain-containing protein Mmi1 and targets certain RNA transcripts to nuclear foci for nuclear retention and decay. Here we show that YTHDC1 and YTHDC2, two YTH domain-containing proteins that bind to N-6-methyladenosine (m6A) modified RNAs, interact with ZFC3H1 and U1-70K, and are required for the nuclear retention of mRNAs with intact 5'SS motifs. Disruption of m6A deposition inhibits both the nuclear retention of these transcripts and their accumulation in YTHDC1-enriched foci that are adjacent to nuclear speckles. Endogenous RNAs with intact 5'SS motifs, such as intronic poly-adenylated transcripts, tend to be m6A-modified at low levels. Thus, the m6A modification acts on a conserved quality control mechanism that targets misprocessed mRNAs for nuclear retention and decay.

DOI: https://doi.org/10.26508/lsa.202403142
Cell Chem Biol. 2024 Nov 25. pii: S2451-9456(24)00459-8. [Epub ahead of print]

β-hydroxybutyrate is a metabolic regulator of proteostasis in the aged and Alzheimer disease brain.

Sidharth S Madhavan, Stephanie Roa Diaz, Sawyer Peralta, Mitsunori Nomura, Christina D King, Kaya E Ceyhan, Anwen Lin, Dipa Bhaumik, Anna C Foulger, Samah Shah, Thanh Blade, Wyatt Gray, Manish Chamoli, Brenda Eap, Oishika Panda, Diego Diaz, Thelma Y Garcia, Brianna J Stubbs, Scott M Ulrich, Gordon J Lithgow, Birgit Schilling, Eric Verdin, Asish R Chaudhuri, John C Newman.

  Loss of proteostasis is a hallmark of aging and Alzheimer disease (AD). We identify β-hydroxybutyrate (βHB), a ketone body, as a regulator of protein solubility. βHB primarily provides ATP substrate during periods of reduced glucose availability, and regulates other cellular processes through protein interactions. We demonstrate βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester. This mechanism is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity. We generate libraries of the βHB-induced protein insolublome using mass spectrometry proteomics, and identify common protein domains and upstream regulators. We show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain. These data indicate a metabolically regulated mechanism of proteostasis relevant to aging and AD.

Keywords:  Alzheimer disease; aging; ketone body; neurodegenerative disease; proteostasis

DOI:  https://doi.org/10.1016/j.chembiol.2024.11.001
Mol Cell. 2024 Nov 25. pii: S1097-2765(24)00908-0. [Epub ahead of print]

Structural dynamics of human ribosomes in situ reconstructed by exhaustive high-resolution template matching.

J Peter Rickgauer, Heejun Choi, Andrew S Moore, Winfried Denk, Jennifer Lippincott-Schwartz.

  Protein synthesis is central to life and requires the ribosome, which catalyzes the stepwise addition of amino acids to a polypeptide chain by undergoing a sequence of structural transformations. Here, we employed high-resolution template matching (HRTM) on cryoelectron microscopy (cryo-EM) images of directly cryofixed living cells to obtain a set of ribosomal configurations covering the entire elongation cycle, with each configuration occurring at its native abundance. HRTM's position and orientation precision and ability to detect small targets (∼300 kDa) made it possible to order these configurations along the reaction coordinate and to reconstruct molecular features of any configuration along the elongation cycle. Visualizing the cycle's structural dynamics by combining a sequence of >40 reconstructions into a 3D movie readily revealed component and ligand movements, some of them surprising, such as spring-like intramolecular motion, providing clues about the molecular mechanisms involved in some still mysterious steps during chain elongation.

Keywords:  HRTM; elongation cycle; in situ cryo-EM; molecular motion; protein translation; ribosome dynamics; structural biology

DOI:  https://doi.org/10.1016/j.molcel.2024.11.003
Nat Commun. 2024 Dec 03. 15(1): 10536

CRL3Keap1 E3 ligase facilitates ubiquitin-mediated degradation of oncogenic SRX to suppress colorectal cancer progression.

Feng Zhu, Liangshan Li, Yuanyuan Chen, Yongfu Pan, Wenjuan Zhang, Lihui Li, Lili Cai, Xiaoxue Zhao, Hu Zhao, Shiwen Wang, Lijun Jia.

The antioxidant protein sulfiredoxin-1 (SRX) is an oncogenic factor that promotes tumor progression, but the regulatory mechanism underlying SRX degradation remains to be understood. Herein, we report that Keap1, the substrate-specific adapter of CRL3 complex, specifically binds and promotes the ubiquitin-mediated degradation of SRX at residue K61. Keap1 knockdown accumulates SRX, which in turn facilitates colorectal cancer (CRC) metastasis by activating the activator protein-1/matrix metalloproteinase 9 (AP-1/MMP9) pathway. CRC-associated Keap1 mutants within the BACK domain lose the capability to ubiquitinate SRX and instead promote CRC metastasis. Moreover, inactivation of Keap1 facilitates CRC tumorigenesis and metastasis in mouse models of tumor xenograft due to SRX accumulation. Clinical sample analysis reveals that Keap1 is downregulated while SRX is overexpressed in CRC, which correlates with poor prognosis. Our findings elucidate a mechanism by which CRL3Keap1 ubiquitin ligase degrades SRX to suppress CRC progression, indicating that the Keap1-SRX axis will guide the targeted therapy towards CRC.

DOI: https://doi.org/10.1038/s41467-024-54919-2
PLoS Biol. 2024 Dec 02. 22(12): e3002942

Intracellular polarization of RNAs and proteins in the human small intestinal epithelium.

Roy Novoselsky, Yotam Harnik, Oran Yakubovsky, Corine Katina, Yishai Levin, Keren Bahar Halpern, Niv Pencovich, Ido Nachmany, Shalev Itzkovitz.

The intestinal epithelium is a polarized monolayer of cells, with an apical side facing the lumen and a basal side facing the blood stream. In mice, both proteins and mRNAs have been shown to exhibit global basal-apical polarization; however, polarization in the human intestine has not been systematically explored. Here, we employed laser-capture microdissection to isolate apical and basal epithelial segments from intestinal tissues of 8 individuals and performed RNA sequencing and mass-spectrometry proteomics. We find a substantial polarization of mRNA molecules that largely overlaps polarization patterns observed in mice. This mRNA polarization remains consistent across different zones of the intestinal villi and is generally correlated with the polarization of proteins. Our protein analysis exposes streamlined intracellular nutrient transport and processing and reveals that mitochondria and ribosomes are less polarized in humans compared to mice. Our study provides a resource for understanding human intestinal epithelial biology.

DOI: https://doi.org/10.1371/journal.pbio.3002942
Nature. 2024 Dec 04.

Tracking transcription-translation coupling in real time.

Nusrat Shahin Qureshi, Olivier Duss.

A central question in biology is how macromolecular machines function cooperatively. In bacteria, transcription and translation occur in the same cellular compartment, and can be physically and functionally coupled1-4. Although high-resolution structures of the ribosome-RNA polymerase (RNAP) complex have provided initial mechanistic insights into the coupling process5-10, we lack knowledge of how these structural snapshots are placed along a dynamic reaction trajectory. Here we reconstitute a complete and active transcription-translation system and develop multi-colour single-molecule fluorescence microscopy experiments to directly and simultaneously track transcription elongation, translation elongation and the physical and functional coupling between the ribosome and the RNAP in real time. Our data show that physical coupling between ribosome and RNAP can occur over hundreds of nucleotides of intervening mRNA by mRNA looping, a process facilitated by NusG. We detect active transcription elongation during mRNA looping and show that NusA-paused RNAPs can be activated by the ribosome by long-range physical coupling. Conversely, the ribosome slows down while colliding with the RNAP. We hereby provide an alternative explanation for how the ribosome can efficiently rescue RNAP from frequent pausing without requiring collisions by a closely trailing ribosome. Overall, our dynamic data mechanistically highlight an example of how two central macromolecular machineries, the ribosome and RNAP, can physically and functionally cooperate to optimize gene expression.

DOI: https://doi.org/10.1038/s41586-024-08308-w
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2409973121

Sequence complexity and monomer rigidity control the morphologies and aging dynamics of protein aggregates.

Ryota Takaki, D Thirumalai.

  Understanding the biophysical basis of protein aggregation is important in biology because of the potential link to several misfolding diseases. Although experiments have shown that protein aggregates adopt a variety of morphologies, the dynamics of their formation are less well characterized. Here, we introduce a minimal model to explore the dependence of the aggregation dynamics on the structural and sequence features of the monomers. Using simulations, we demonstrate that sequence complexity (codified in terms of word entropy) and monomer rigidity profoundly influence the dynamics and morphology of the aggregates. Flexible monomers with low sequence complexity (corresponding to repeat sequences) form liquid-like droplets that exhibit ergodic behavior. Strikingly, these aggregates abruptly transition to more ordered structures, reminiscent of amyloid fibrils, when the monomer rigidity is increased. In contrast, aggregates resulting from monomers with high sequence complexity are amorphous and display nonergodic glassy dynamics. The heterogeneous dynamics of the low and high-complexity sequences follow stretched exponential kinetics, which is one of the characteristics of glassy dynamics. Importantly, at nonzero values of the bending rigidities, the aggregates age with the relaxation times that increase with the waiting time. Informed by these findings, we provide insights into aging dynamics in protein condensates and contrast the behavior with the dynamics expected in RNA repeat sequences. Our findings underscore the influence of the monomer characteristics in shaping the morphology and dynamics of protein aggregates, thus providing a foundation for deciphering the general rules governing the behavior of protein condensates.

Keywords:  biological condensates; dynamic heterogeneity; ergodicity breaking; polymer glass; slow dynamics

DOI:  https://doi.org/10.1073/pnas.2409973121
Nat Biotechnol. 2024 Dec 03.

Engineered platelets as targeted protein degraders and application to breast cancer models.

Yu Chen, Samira Pal, Wen Li, Fengyuan Liu, Sichen Yuan, Quanyin Hu.

Clinical application of chimeric molecules for targeted protein degradation has been limited by unfavorable drug-like properties and biosafety concerns arising from nonspecific biodistribution after systemic administration. Here we develop a method to engineer platelets for degradation of either intracellular or extracellular proteins of interest (POIs) in vivo by covalently labeling heat shock protein 90 (HSP90) in platelets with a POI ligand. The degrader platelets (DePLTs) target wound areas and undergo activation. Depending on the tethered POI ligand and transport mechanism of the prelabeled HSP90, activated DePLTs can mediate targeted protein degradation in the target cell through the ubiquitin-proteasome machinery or the lysosome. HSP90 packaged into platelet-derived microparticles uses the ubiquitin-proteasome system to degrade intracellular POIs, whereas released free HSP90 redirects extracellular POIs to lysosomal degradation. In postsurgical breast cancer mouse models, DePLTs engineered with corresponding POI ligands effectively degrade intracellular bromodomain-containing protein 4 or extracellular programmed cell death ligand 1, thereby suppressing cancer recurrence or metastasis.

DOI: https://doi.org/10.1038/s41587-024-02494-8
Cell Commun Signal. 2024 Dec 02. 22(1): 577

The Yin and Yang of hsa-miR-1244 expression levels during activation of the UPR control cell fate.

Paulina Czechowicz, Magdalena Gebert, Sylwia Bartoszewska, Leszek Kalinowski, James F Collawn, Rafal Bartoszewski.

  Regulation of endoplasmic reticulum (ER) homeostasis plays a critical role in maintaining cell survival. When ER stress occurs, a network of three pathways called the unfolded protein response (UPR) is activated to reestablish homeostasis. While it is known that there is cross-talk between these pathways, how this complex network is regulated is not entirely clear. Using human cancer and non-cancer cell lines, two different genome-wide approaches, and two different ER stress models, we searched for miRNAs that were decreased during the UPR and surprisingly found only one, miR-1244, that was found under all these conditions. We also verified that ER-stress related downregulation of miR-1244 expression occurred with 5 different ER stressors and was confirmed in another human cell line (HeLa S3). These analyses demonstrated that the outcome of this reduction during ER stress supported both IRE1 signaling and elevated BIP expression. Further analysis using inhibitors specific for IRE1, ATF6, and PERK also revealed that this miRNA is impacted by all three pathways of the UPR. This is the first example of a complex mechanism by which this miRNA serves as a regulatory check point for all 3 pathways that is switched off during UPR activation. In summary, the results indicate that ER stress reduction of miR-1244 expression contributes to the pro-survival arm of UPR.

Keywords:  Cell fate decisions; ER stress; UPR; miRNA; microRNA

DOI:  https://doi.org/10.1186/s12964-024-01967-2
Oncogene. 2024 Dec 03.

Discovery of a molecular glue for EGFR degradation.

Hairui Wang, Hui Wang, Rui Wang, Yuanzhen Li, Zhipeng Wang, Wenshen Zhou, Li Deng, Xiyin Li, Li Zou, Qin Yang, Ren Lai, Xiaowei Qi, Jianyun Nie, Baowei Jiao.

Aberrant expression of epidermal growth factor receptor (EGFR) plays a critical role in the pathogenesis of various tumors, potentially representing a target for therapeutic intervention. Nonetheless, EGFR remains a challenging protein to target pharmacologically in triple-negative breast cancer (TNBC). An emerging approach to address the removal of such proteins is the application of molecular glue (MG) degraders. These compounds facilitate protein-protein interactions between a target protein and an E3-ubiquitin ligase, subsequently leading to protein degradation. Herein, we identified a new MG (CDDO-Me, C-28 methyl ester of 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acid), which orchestrated binding between EGFR and KEAP1 (an E3-ubiquitin ligase adapter), thereby initiating the ubiquitination and degradation of EGFR. CDDO-Me directly interacted with the tyrosine kinase (TK) domain of EGFR, resulting in its degradation via an autophagy-dependent lysosomal pathway. Knockdown of KEAP1 decreased the degradation of EGFR by reducing its K63-linked ubiquitination, leading to diminished EGFR colocalization in autophagosomes and lysosomes. Notably, CDDO-Me attenuates TNBC progression by accelerating EGFR degradation in cell-derived xenografts and patient-derived organoid models, highlighting its clinical application potential. Consequently, induction of EGFR degradation through MG degraders represents a viable therapeutic strategy for TNBC.

DOI: https://doi.org/10.1038/s41388-024-03241-8
Nature. 2024 Dec 04.

Synthetic GPCRs for programmable sensing and control of cell behaviour.

Nicholas A Kalogriopoulos, Reika Tei, Yuqi Yan, Peter M Klein, Matthew Ravalin, Bo Cai, Ivan Soltesz, Yulong Li, Alice Ting.

Synthetic receptors that mediate antigen-dependent cell responses are transforming therapeutics, drug discovery and basic research1,2. However, established technologies such as chimeric antigen receptors3 can only detect immobilized antigens, have limited output scope and lack built-in drug control3-7. Here we engineer synthetic G-protein-coupled receptors (GPCRs) that are capable of driving a wide range of native or non-native cellular processes in response to a user-defined antigen. We achieve modular antigen gating by engineering and fusing a conditional auto-inhibitory domain onto GPCR scaffolds. Antigen binding to a fused nanobody relieves auto-inhibition and enables receptor activation by drug, thus generating programmable antigen-gated G-protein-coupled engineered receptors (PAGERs). We create PAGERs that are responsive to more than a dozen biologically and therapeutically important soluble and cell-surface antigens in a single step from corresponding nanobody binders. Different PAGER scaffolds allow antigen binding to drive transgene expression, real-time fluorescence or endogenous G-protein activation, enabling control of diverse cellular functions. We demonstrate multiple applications of PAGER, including induction of T cell migration along a soluble antigen gradient, control of macrophage differentiation, secretion of therapeutic antibodies and inhibition of neuronal activity in mouse brain slices. Owing to its modular design and generalizability, we expect PAGERs to have broad utility in discovery and translational science.

DOI: https://doi.org/10.1038/s41586-024-08282-3
Nat Commun. 2024 Dec 01. 15(1): 10447

Protein engineering using variational free energy approximation.

Evgenii Lobzaev, Michael A Herrera, Martyna Kasprzyk, Giovanni Stracquadanio.

Engineering proteins is a challenging task requiring the exploration of a vast design space. Traditionally, this is achieved using Directed Evolution (DE), which is a laborious process. Generative deep learning, instead, can learn biological features of functional proteins from sequence and structural datasets and return novel variants. However, most models do not generate thermodynamically stable proteins, thus leading to many non-functional variants. Here we propose a model called PRotein Engineering by Variational frEe eNergy approximaTion (PREVENT), which generates stable and functional variants by learning the sequence and thermodynamic landscape of a protein. We evaluate PREVENT by designing 40 variants of the conditionally essential E. coli phosphotransferase N-acetyl-L-glutamate kinase (EcNAGK). We find 85% of the variants to be functional, with 55% of them showing similar growth rate compared to the wildtype enzyme, despite harbouring up to 9 mutations. Our results support a new approach that can significantly accelerate protein engineering.

DOI: https://doi.org/10.1038/s41467-024-54814-w
Sci Signal. 2024 Dec 03. 17(865): eadk7971

ChREBP-mediated up-regulation of Them1 coordinates thermogenesis with glycolysis and lipogenesis in response to chronic stress.

Xu Xu, Arturo Mendoza, Christopher S Krumm, Shi Su, Mariana Acuña, Curtis J Bare, Corey D Holman, Marissa Cortopassi, Hayley T Nicholls, Vincent Dartigue, Anthony N Hollenberg, Ann-Hwee Lee, Susan J Hagen, David E Cohen.

Activation of thermogenic brown adipose tissue (BAT) and inducible beige adipose tissue (BeAT) is triggered by environmental or metabolic stimuli, including cold ambient temperatures and nutrient stress. Thioesterase superfamily member 1 (Them1), a long-chain fatty acyl-CoA thioesterase that is enriched in BAT, suppresses acute cold-induced thermogenesis. Here, we demonstrate that Them1 expression was induced in BAT and BeAT by the carbohydrate response element binding protein (ChREBP) in response to chronic cold exposure or to the activation of the integrated stress response (ISR) by nutrient excess. Under either condition, Them1 suppressed energy expenditure. Consequently, mice lacking Them1 in BAT and BeAT exhibited resistance to obesity and glucose intolerance induced by feeding with a high-fat diet. During chronic cold exposure or ISR activation, Them1 accumulated in the nucleus, where it interacted with ChREBP and reduced the expression of its target genes, including those encoding enzymes that mediate glycolysis and de novo lipogenesis. These findings demonstrate that in response to chronic cold- or nutrient-induced stress, the induction of Them1 by ChREBP limits thermogenesis while coordinately reducing glucose utilization and lipid biosynthesis through its distinct cytoplasmic and nuclear activities. Targeted inhibition of Them1 could be a potential therapeutic approach to increase the activity of BAT and BeAT to enhance energy expenditure in the management of obesity-associated metabolic disorders.

DOI: https://doi.org/10.1126/scisignal.adk7971
Cell Mol Gastroenterol Hepatol. 2024 Dec 03. pii: S2352-345X(24)00189-9. [Epub ahead of print] 101434

Chronic gastroesophageal reflux dysregulates proteostasis in esophageal epithelial cells.

Kodisundaram Paulrasu, Ravindran Caspa Gokulan, Wael El-Rifai, Zhibin Chen, Jianwen Que, Timothy C Wang, Olivier G Boutaud, Karoline Briegel, Sergey I Dikalov, Monica T Garcia-Buitrago, Alexander I Zaika.

   BACKGROUND AND AIMS: Gastroesophageal reflux disease (GERD) is a common digestive disorder that is characterized by esophageal tissue damage produced by exposure of the esophageal lining to the gastric refluxate. GERD can raise the risk of multiple serious complications including esophageal tumors. At the molecular levels, GERD-affected tissues are characterized by strong oxidative stress and the formation of reactive isolevuglandins (isoLGs). These products of lipid peroxidation rapidly interact with cellular proteins forming protein adducts. Here, we investigated the interrelationship between isoLG adduction and aggregation of cellular proteins.
METHODS: Protein misfolding and aggregation were analyzed using multiple protein misfolding and aggregation assays. Pathological consequences of protein adduction and aggregation were studied using human and murine esophageal tissues. Surgical model of esophageal reflux injury and L2-IL1β transgenic mice were employed to investigate the mechanisms of protein misfolding and aggregation.
RESULTS: Our studies demonstrate that gastroesophageal reflux causes protein misfolding and aggregation that is associated with severity of GERD. Dysregulation of proteostasis induces ferroptotic cell death and is mediated by modification of cellular proteins with reactive isoLGs that can be prevented by isoLG scavengers.
CONCLUSIONS: GERD causes dysregulation of cellular proteostasis, accumulation of isoLG protein adducts, misfolded and aggregated proteins that promote ferroptotic cell death. Taken together, this study suggests that GERD has similarities to other known pathological conditions that are characterized by protein misfolding and aggregation.

Keywords:  GERD; lipid peroxidation; oxidative stress; proteostasis

DOI:  https://doi.org/10.1016/j.jcmgh.2024.101434
J Cell Mol Med. 2024 Dec;28(23): e70261

Cytoplasmic Aggregates of Splicing Factor Proline-Glutamine Rich Disrupt Nucleocytoplasmic Transport and Induce Persistent Stress Granules.

Zicong Huang, Hanbin Zhang, Chuyu Huang, Runduan Yi, Xiaoyuan Zhang, Ke Ma, Wei Huang, Qingqing Wu, Yuge Zhuang, Jinsheng Liu, Wenyuan Liu, Yunhui Guo, Xiangjin Kang, Xiao Hu, Xiaochun Bai, Zhenguo Chen.

  Splicing factor proline-glutamine rich (SFPQ), a multifunctional RNA-binding protein (RBP), shows cytoplasmic colocalisation with stress granule (SG) markers; however, the causative relationship and mechanism underlying this coalescence of SFPQ aggregates and SGs remain unclear. In this study, we demonstrate that SFPQ lacking its nuclear localisation sequence spontaneously forms cytoplasmic aggregates that abnormally incorporate immature RNA and induce persistent SGs. mRNA profiling showed that SFPQ mislocalisation induced extensive changes in RNA processing, with a subset of alternatively spliced transcripts associated with nucleocytoplasmic transport. Notably, these altered transporters were sequestered into SFPQ aggregates, constituting aberrant protein-RNA complexes. Importantly, suppression of SG nucleation could not block cytoplasmic SFPQ aggregation with immature RNA and nucleocytoplasmic transporters, both of which, however, were moderately ameliorated by the inhibition of alternative splicing or nuclear export. Our results unveil the physiopathological role and mechanism for mislocalised SFPQ in the RNA metabolism, nucleocytoplasmic transport and pathological SGs.

Keywords:  RNA metabolism; SFPQ; cytoplasmic aggregate; nucleocytoplasmic transport; stress granule

DOI:  https://doi.org/10.1111/jcmm.70261
Nat Commun. 2024 Dec 04. 15(1): 10592

Yin Yang 1 and guanine quadruplexes protect dopaminergic neurons from cellular stress via transmissive dormancy.

Kielen R Zuurbier, Rene Solano Fonseca, Sonja L B Arneaud, Jordan M Wall, Juhee Kim, Lexus Tatge, Gupse Otuzoglu, Sofia Bali, Patrick Metang, Peter M Douglas.

Neurons deploy diverse adaptive strategies to ensure survival and neurotransmission amid cellular stress. When these adaptive pathways are overwhelmed, functional impairment or neurodegeneration follows. Here we show that stressed neurons actively induce a state of transmissive dormancy as a protective measure. Extending observations of neurotrauma in C. elegans and mice, human dopaminergic neurons capable of surviving severe cellular challenges both decrease spontaneous activity and modulate dopamine homeostasis through the transcriptional regulator Yin Yang 1 (YY1). To bolster stress resilience and mitigate dopamine toxicity, YY1 increases expression of the vesicular monoamine transporter 2, vMAT2, while coordinately inhibiting dopamine synthesis through stabilization of a guanine quadruplex in intron 10 of tyrosine hydroxylase, TH. This dopaminergic stress response has the potential to cause circuit inactivation, yet safeguards neurons by minimizing the toxic accumulation of cytosolic dopamine and inducing a state of neuronal dormancy. In essence, neurons appear to actively prioritize viability over functionality.

DOI: https://doi.org/10.1038/s41467-024-54958-9
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2408114121

Intricate ribosome composition and translational reprogramming in epithelial-mesenchymal transition.

Chloé Morin, Agnès Baudin-Baillieu, Flora Nguyen Van Long, Caroline Isaac, Laure Bidou, Hugo Arbes, Pauline François, Roxane M Pommier, Annie Adrait, Akari Saku, Stephanie Gran-Ruaz, Camélia Machkouri, Christophe Vanbelle, Romain Morichon, Mathieu Boissan, Frédéric Catez, Anthony Ferrari, Anne-Pierre Morel, Yohann Couté, Sophie Chat, Emmanuel Giudice, Reynald Gillet, Alain Puisieux, Caroline Moyret-Lalle, Jean-Jacques Diaz, Olivier Namy, Virginie Marcel.

  Epithelial-mesenchymal transition (EMT) involves profound changes in cell morphology, driven by transcriptional and epigenetic reprogramming. However, evidence suggests that translation and ribosome composition also play key roles in establishing pathophysiological phenotypes. Using genome-wide analyses, we reported significant rearrangement of the translational landscape and machinery during EMT. Specifically, a cell line overexpressing the EMT transcription factor ZEB1 displayed alterations in translational reprogramming and fidelity. Furthermore, using riboproteomics, we unveiled an increased level of the ribosomal protein RPL36A in mesenchymal ribosomes, indicating precise tuning of ribosome composition. Remarkably, RPL36A overexpression alone was sufficient to trigger the acquisition of mesenchymal features, including a switch in the molecular pattern, cell morphology, and behavior, demonstrating its pivotal role in EMT. These findings underline the importance of translational reprogramming and fine-tuning of ribosome composition in EMT.

Keywords:  EMT; RPL36A; ZEB1; ribosome; translation

DOI:  https://doi.org/10.1073/pnas.2408114121