bims-proteo 2024-11-10 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–11–10
29 papers selected by
Eric Chevet, INSERM

Threonine-rich carboxyl-terminal extension drives aggregation of stalled polypeptides.
The structure and function of P5A-ATPases.
The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation.
Selective degradation of multimeric proteins by TRIM21-based molecular glue and PROTAC degraders.
Homeostasis control in health and disease by the unfolded protein response.
AMFR-mediated Flavivirus NS2A ubiquitination subverts ER-phagy to augment viral pathogenicity.
mTORC1 activity licenses its own release from the lysosomal surface.
Perturbations in eIF3 subunit stoichiometry alter expression of ribosomal proteins and key components of the MAPK signaling pathways.
The Arabidopsis E3 ubiquitin ligase DOA10A promotes localization of abscisic acid (ABA) receptors to the membrane through mono-ubiquitination in ABA signaling.
Identification of a non-canonical function of prefoldin subunit 5 in proteasome assembly.
Next steps for targeted protein degradation.
Ca2+ tunneling architecture and function are important for secretion.
A switch from lysosomal degradation to secretory autophagy initiates osteogenic bone metastasis in prostate cancer.
ER Oxidoreductin 1-Like Activity of Cyclic Diselenides Drives Protein Disulfide Isomerase in an Electron Relay System.
A mechanism for hypoxia-induced inflammatory cell death in cancer.
Augmenting Protein Degradation Capacity of PROTAC through Energy Metabolism Regulation and Targeted Drug Delivery.
Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress.
LMAN1 serves as a cargo receptor for thrombopoietin.
Deciphering the RNA-binding protein network during endosomal mRNA transport.
An outcome-defining role for the triple-helical domain in regulating collagen-I assembly.
Discovery of CRBN-Dependent WEE1 Molecular Glue Degraders from a Multicomponent Combinatorial Library.
CRBN-PROTACs in Cancer Therapy: From Mechanistic Insights to Clinical Applications.
Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion.
Extracellular proximal interaction profiling by cell surface-targeted TurboID reveals LDLR as a partner of liganded EGFR.
Protocol for mapping differential protein-protein interaction networks using affinity purification-mass spectrometry.
Targeting the HECTD3-p62 axis increases the radiosensitivity of triple negative breast cancer cells.
Endosomes serve as signaling platforms for RIG-I ubiquitination and activation.
The HSP90/R2TP quaternary chaperone scaffolds assembly of the TSC complex.
The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay.

Mol Cell. 2024 Nov 01. pii: S1097-2765(24)00834-7. [Epub ahead of print]

Threonine-rich carboxyl-terminal extension drives aggregation of stalled polypeptides.

Weili Denyse Chang, Mi-Jeong Yoon, Kian Hua Yeo, Young-Jun Choe.

  Ribosomes translating damaged mRNAs may stall and prematurely split into their large and small subunits. The split large ribosome subunits can continue elongating stalled polypeptides. In yeast, this mRNA-independent translation appends the C-terminal alanine/threonine tail (CAT tail) to stalled polypeptides. If not degraded by the ribosome-associated quality control (RQC), CAT-tailed stalled polypeptides form aggregates. How the CAT tail, a low-complexity region composed of alanine and threonine, drives protein aggregation remains unknown. In this study, we demonstrate that C-terminal polythreonine or threonine-enriched tails form detergent-resistant aggregates. These aggregates exhibit a robust seeding effect on shorter tails with lower threonine content, elucidating how heterogeneous CAT tails co-aggregate. Polythreonine aggregates sequester molecular chaperones, disturbing proteostasis and provoking the heat shock response. Furthermore, polythreonine cross-seeds detergent-resistant polyserine aggregation, indicating structural similarity between the two aggregates. This study identifies polythreonine and polyserine as a distinct group of aggregation-prone protein motifs.

Keywords:  CAT tail; RQC; chaperone; heat shock response; molecular chaperone; polyQ; polyserine; polyserine aggregation; polythreonine; polythreonine aggregation; proteostasis; ribosome stalling

DOI:  https://doi.org/10.1016/j.molcel.2024.10.011
Nat Commun. 2024 Nov 06. 15(1): 9605

The structure and function of P5A-ATPases.

Ping Li, Viktoria Bågenholm, Per Hägglund, Karin Lindkvist-Petersson, Kaituo Wang, Pontus Gourdon.

Endoplasmic reticulum (ER) membrane resident P5A-ATPases broadly affect protein biogenesis and quality control, and yet their molecular function remains debated. Here, we report cryo-EM structures of a P5A-ATPase, CtSpf1, covering multiple transport intermediates of the E1 → E1-ATP → E1P-ADP → E1P → E2P → E2.Pi → E2 → E1 cycle. In the E2P and E2.Pi states a cleft spans the entire membrane, holding a polypeptide cargo molecule. The cargo includes an ER luminal extension, pinpointed as the C-terminus in the E2.Pi state, which reenters the membrane in E2P. The E1 structure harbors a cytosol-facing cavity that is blocked by an insertion we refer to as the Plug-domain. The Plug-domain is nestled to key ATPase features and is displaced in the E1P-ADP and E1P states. Collectively, our findings are compatible with a broad range of proteins as cargo, with the P5A-ATPases serving a role in membrane removal of helices, although insertion/secretion cannot be excluded, as well as with a mechanistic role of the Plug-domain.

DOI: https://doi.org/10.1038/s41467-024-53757-6
EMBO Rep. 2024 Nov 06.

The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation.

Kaikai Yu, Guan M Wang, Shiny Shengzhen Guo, Florian Bassermann, Reinhard Fässler.

  The functions of integrins are tightly regulated via multiple mechanisms including trafficking and degradation. Integrins are repeatedly internalized, routed into the endosomal system and either degraded by the lysosome or recycled back to the plasma membrane. The ubiquitin system dictates whether internalized proteins are degraded or recycled. Here, we use a genetic screen and proximity-dependent biotin identification to identify deubiquitinase(s) that control integrin surface levels. We find that a ternary deubiquitinating complex, comprised of USP12 (or the homologous USP46), WDR48 and WDR20, stabilizes β1 integrin (Itgb1) by preventing ESCRT-mediated lysosomal degradation. Mechanistically, the USP12/46-WDR48-WDR20 complex removes ubiquitin from the cytoplasmic tail of internalized Itgb1 in early endosomes, which in turn prevents ESCRT-mediated sorting and Itgb1 degradation.

Keywords:  DUB; ESCRT; Integrin; USP12/USP46; Ubiquitination

DOI:  https://doi.org/10.1038/s44319-024-00300-9
Cell. 2024 Oct 30. pii: S0092-8674(24)01197-8. [Epub ahead of print]

Selective degradation of multimeric proteins by TRIM21-based molecular glue and PROTAC degraders.

Panrui Lu, Yalong Cheng, Lei Xue, Xintong Ren, Xilong Xu, Chenglong Chen, Longzhi Cao, Jiaojiao Li, Qingcui Wu, Shan Sun, Junjie Hou, Wei Jia, Wei Wang, Yan Ma, Zhaodi Jiang, Chao Li, Xiangbing Qi, Niu Huang, Ting Han.

  Targeted protein degradation (TPD) utilizes molecular glues or proteolysis-targeting chimeras (PROTACs) to eliminate disease-causing proteins by promoting their interaction with E3 ubiquitin ligases. Current TPD approaches are limited by reliance on a small number of constitutively active E3 ubiquitin ligases. Here, we report that (S)-ACE-OH, a metabolite of the antipsychotic drug acepromazine, acts as a molecular glue to induce an interaction between the E3 ubiquitin ligase TRIM21 and the nucleoporin NUP98, leading to the degradation of nuclear pore proteins and disruption of nucleocytoplasmic trafficking. Functionalization of acepromazine into PROTACs enabled selective degradation of multimeric proteins, such as those within biomolecular condensates, while sparing monomeric proteins. This selectivity is consistent with the requirement of substrate-induced clustering for TRIM21 activation. As aberrant protein assemblies cause diseases such as autoimmunity, neurodegeneration, and cancer, our findings highlight the potential of TRIM21-based multimer-selective degraders as a strategy to tackle the direct causes of these diseases.

Keywords:  E3 ubiquitin ligase; PROTAC; TRIM21; biomolecular condensate; molecular glue; multimeric proteins; nuclear pore complex; targeted protein degradation

DOI:  https://doi.org/10.1016/j.cell.2024.10.015
Nat Rev Mol Cell Biol. 2024 Nov 05.

Homeostasis control in health and disease by the unfolded protein response.

Diego Acosta-Alvear, Jonathan M Harnoss, Peter Walter, Avi Ashkenazi.

Cells rely on the endoplasmic reticulum (ER) to fold and assemble newly synthesized transmembrane and secretory proteins - essential for cellular structure-function and for both intracellular and intercellular communication. To ensure the operative fidelity of the ER, eukaryotic cells leverage the unfolded protein response (UPR) - a stress-sensing and signalling network that maintains homeostasis by rebalancing the biosynthetic capacity of the ER according to need. The metazoan UPR can also redirect signalling from cytoprotective adaptation to programmed cell death if homeostasis restoration fails. As such, the UPR benefits multicellular organisms by preserving optimally functioning cells while removing damaged ones. Nevertheless, dysregulation of the UPR can be harmful. In this Review, we discuss the UPR and its regulatory processes as a paradigm in health and disease. We highlight important recent advances in molecular and mechanistic understanding of the UPR that enable greater precision in designing and developing innovative strategies to harness its potential for therapeutic gain. We underscore the rheostatic character of the UPR, its contextual nature and critical open questions for its further elucidation.

DOI: https://doi.org/10.1038/s41580-024-00794-0
Nat Commun. 2024 Nov 06. 15(1): 9578

AMFR-mediated Flavivirus NS2A ubiquitination subverts ER-phagy to augment viral pathogenicity.

Linliang Zhang, Hongyun Wang, Chao Han, Qi Dong, Jie Yan, Weiwei Guo, Chao Shan, Wen Zhao, Pu Chen, Rui Huang, Ying Wu, Yu Chen, Yali Qin, Mingzhou Chen.

Flaviviruses strategically utilize the endoplasmic reticulum (ER) in their replication cycles. However, the role of ER autophagy (ER-phagy) in viral replication process remains poorly understood. Here, we reveal that prolonged Zika virus (ZIKV) infection results from the degradation of ER-phagy receptor FAM134B, facilitated by viral NS2A protein. Mechanistically, ER-localized NS2A undergoes K48-linked polyubiquitination at lysine (K) 56 by E3 ligase AMFR. Ubiquitinated NS2A binds to FAM134B and AMFR orchestrates the degradation of NS2A-FAM134B complexes. AMFR-catalyzed NS2A ubiquitination not only targets FAM134B degradation but also hinders the FAM134B-AMFR axis. Notably, a recombinant ZIKV mutant (ZIKV-NS2AK56R), lacking ubiquitination and ER-phagy inhibition, exhibits attenuation in ZIKV-induced microcephalic phenotypes in human brain organoids and replicates less efficiently, resulting in weakened pathogenesis in mouse models. In this work, our mechanistic insights propose that flaviviruses manipulate ER-phagy to modulate ER turnover, driving viral infection. Furthermore, AMFR-mediated flavivirus NS2A ubiquitination emerges as a potential determinant of viral pathogenecity.

DOI: https://doi.org/10.1038/s41467-024-54010-w
Mol Cell. 2024 Oct 25. pii: S1097-2765(24)00831-1. [Epub ahead of print]

mTORC1 activity licenses its own release from the lysosomal surface.

Aishwarya Acharya, Constantinos Demetriades.

  Nutrient signaling converges on mTORC1, which, in turn, orchestrates a physiological cellular response. A key determinant of mTORC1 activity is its shuttling between the lysosomal surface and the cytoplasm, with nutrients promoting its recruitment to lysosomes by the Rag GTPases. Active mTORC1 regulates various cellular functions by phosphorylating distinct substrates at different subcellular locations. Importantly, how mTORC1 that is activated on lysosomes is released to meet its non-lysosomal targets and whether mTORC1 activity itself impacts its localization remain unclear. Here, we show that, in human cells, mTORC1 inhibition prevents its release from lysosomes, even under starvation conditions, which is accompanied by elevated and sustained phosphorylation of its lysosomal substrate TFEB. Mechanistically, "inactive" mTORC1 causes persistent Rag activation, underlining its release as another process actively mediated via the Rags. In sum, we describe a mechanism by which mTORC1 controls its own localization, likely to prevent futile cycling on and off lysosomes.

Keywords:  GATOR1; Rag GTPases; Rheb; TFE3; TFEB; Torin1; lysosomes; mTORC1; rapamycin

DOI:  https://doi.org/10.1016/j.molcel.2024.10.008
Elife. 2024 Nov 04. pii: RP95846. [Epub ahead of print]13

Perturbations in eIF3 subunit stoichiometry alter expression of ribosomal proteins and key components of the MAPK signaling pathways.

Anna Herrmannová, Jan Jelínek, Klára Pospíšilová, Farkas Kerényi, Tomáš Vomastek, Kathleen Watt, Jan Brábek, Mahabub Pasha Mohammad, Susan Wagner, Ivan Topisirovic, Leoš Shivaya Valášek.

  Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the proto-oncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5' UTRs that are implicated in homeostatic adaptation to stress and cancer.

Keywords:  MAPK pathway; eIF3; genetics; genomics; human; ribosomal proteins; ribosome; translation; translational control

DOI:  https://doi.org/10.7554/eLife.95846
New Phytol. 2024 Nov 04.

The Arabidopsis E3 ubiquitin ligase DOA10A promotes localization of abscisic acid (ABA) receptors to the membrane through mono-ubiquitination in ABA signaling.

Cuixia Liu, Qingliang Li, Zhengwei Shen, Ran Xia, Qian Chen, Xiao Li, Yanglin Ding, Shuhua Yang, Giovanna Serino, Qi Xie, Feifei Yu.

  The endoplasmic reticulum-associated degradation (ERAD) system eliminates misfolded and short-lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain-of-function mutant, doa10a-1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono-ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING-type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono-ubiquitination.

Keywords:  ABA signaling; Arabidopsis; DOA10A; E3 ubiquitin ligase; ERAD; mono‐ubiquitination

DOI:  https://doi.org/10.1111/nph.20224
J Mol Biol. 2024 Oct 25. pii: S0022-2836(24)00467-4. [Epub ahead of print] 168838

Identification of a non-canonical function of prefoldin subunit 5 in proteasome assembly.

Somayeh Shahmoradi Ghahe, Krzysztof Drabikowski, Monika Stasiak, Ulrike Topf.

  The prefoldin complex is a heterohexameric, evolutionarily conserved co-chaperone that assists in folding of polypeptides downstream of the protein translation machinery. Loss of prefoldin function leads to impaired solubility of cellular proteins. The degradation of proteins by the proteasome is an integral part of protein homeostasis. Failure of regulated protein degradation can lead to the accumulation of misfolded and defective proteins. We show that prefoldin subunit 5 is required for proteasome activity by contributing to the assembly of the 26S proteasome. In particular, we found that absence of the prefoldin subunit 5 impairs formation of the Rpt ring subcomplex of the proteasome. Concomitant deletion of PFD5 and HSM3, a chaperone for assembly of the ATPase subunits comprising the Rpt ring, exacerbates this effect, suggesting a synergistic relationship between the two factors in proteasome assembly. Thus, our findings reveal a regulatory mechanism wherein prefoldin subunit 5 plays a crucial role in maintaining proteasome integrity, thereby influencing the degradation of proteins.

Keywords:  Chaperone; Hsm3; Pfd5; Prefoldin; Proteasome; Protein quality control; Ubiquitin

DOI:  https://doi.org/10.1016/j.jmb.2024.168838
Cell Chem Biol. 2024 Oct 29. pii: S2451-9456(24)00439-2. [Epub ahead of print]

Next steps for targeted protein degradation.

Mackenzie W Krone, Craig M Crews.

Targeted protein degradation (TPD) has greatly advanced as a therapeutic strategy in the past two decades, and we are on the cusp of rationally designed protein degraders reaching clinical approval. Offering pharmacological advantages relative to occupancy-driven protein inhibition, chemical methods for regulating biomolecular proximity have provided opportunities to tackle disease-related targets that were undruggable. Despite the pre-clinical success of designed degraders and existence of clinical therapies that serendipitously utilize TPD, expansion of the TPD toolbox is necessary to identify and characterize the next generation of molecular degraders. Here we highlight three areas for continued growth in the field that should be prioritized: expansion of TPD platform with greater spatiotemporal precision, increased throughput of degrader synthesis, and optimization of cooperativity in chemically induced protein complexes. The future is bright for TPD in medicine, and we expect that innovative approaches will increase therapeutic applications of proximity-induced pharmacology.

DOI: https://doi.org/10.1016/j.chembiol.2024.10.004
J Cell Biol. 2025 Jan 06. pii: e202402107. [Epub ahead of print]224(1):

Ca2+ tunneling architecture and function are important for secretion.

Raphael J Courjaret, Larry E Wagner, Rahaf R Ammouri, David I Yule, Khaled Machaca.

Ca2+ tunneling requires both store-operated Ca2+ entry (SOCE) and Ca2+ release from the endoplasmic reticulum (ER). Tunneling expands the SOCE microdomain through Ca2+ uptake by SERCA into the ER lumen where it diffuses and is released via IP3 receptors. In this study, using high-resolution imaging, we outline the spatial remodeling of the tunneling machinery (IP3R1; SERCA; PMCA; and Ano1 as an effector) relative to STIM1 in response to store depletion. We show that these modulators redistribute to distinct subdomains laterally at the plasma membrane (PM) and axially within the cortical ER. To functionally define the role of Ca2+ tunneling, we engineered a Ca2+ tunneling attenuator (CaTAr) that blocks tunneling without affecting Ca2+ release or SOCE. CaTAr inhibits Cl- secretion in sweat gland cells and reduces sweating in vivo in mice, showing that Ca2+ tunneling is important physiologically. Collectively our findings argue that Ca2+ tunneling is a fundamental Ca2+ signaling modality.

DOI: https://doi.org/10.1083/jcb.202402107
J Extracell Vesicles. 2024 Nov;13(11): e70002

A switch from lysosomal degradation to secretory autophagy initiates osteogenic bone metastasis in prostate cancer.

Xiaoyu Wei, Mengmeng Liang, Min Deng, Ji Zheng, Fei Luo, Qinyu Ma.

  The identification of both autophagy-related material degradation and unconventional secretion has paved the way for significant breakthroughs linking autophagy to a plethora of physiological processes and disease conditions. However, the mechanisms that coordinate these two pathways remain elusive. Here, we demonstrate that a switch from the lysosomal degradation to a secretory autophagy pathway is governed by protein tyrosine phosphatase 1B (PTP1B, encoded by PTPN1). Dephosphorylation at two tyrosine residues of syntaxin17 (STX17) by PTP1B reduces autophagosome-lysosome fusion while switching the cells to a secretory autophagy pathway. Both PTP1B overexpression and tumour-derived extracellular vesicles (EVs) can activate the secretory autophagy pathway in osteoblasts. Moreover, we demonstrate that osteoblastic LC3+ EVs, generated via the secretory autophagy pathway, are the primary contributor to tumour-associated bone remodelling in prostate cancer. Depletion of tumour-derived EVs secretion or genetic ablation of osteoblastic PTP1B rescues aberrant bone remodelling and lesions, highlighting the relevance between LC3+ EVs and the formation of bone metastatic niche. Our results reveal the significance of tumour-regulated PTP1B in the fate decision of autophagosomes, and propose a role ofLC3+ EVs in shaping the bone metastatic niche.

Keywords:  autophagosome‐lysosome fusion; bone metastatic niche; extracellular vesicles; secretory autophagy; tumour‐associated bone remodelling

DOI:  https://doi.org/10.1002/jev2.70002
Chembiochem. 2024 Nov 06. e202400739

ER Oxidoreductin 1-Like Activity of Cyclic Diselenides Drives Protein Disulfide Isomerase in an Electron Relay System.

Rumi Mikami, Yuya Nishizawa, Yuki Iwata, Shingo Kanemura, Masaki Okumura, Kenta Arai.

  Disulfide formation generally involves a two-electron oxidation reaction between cysteine residues. Additionally, disulfide formation is an essential post-translational modification for the structural maturation of proteins. This oxidative folding is precisely controlled by an electron relay network constructed by protein disulfide isomerase (PDI), with a CGHC sequence as the redox-active site, and its family enzymes. Creating reagents that mimic the functions of these enzymes facilitates folding during chemical protein synthesis. In this study, we aimed to imitate a biological electron relay system using cyclic diselenide compounds as surrogates for endoplasmic reticulum oxidoreductin 1 (Ero1), which is responsible for the re-oxidation of PDI. Oxidized PDI (PDIox) introduces disulfide bonds into substrate proteins, resulting in its conversion to reduced PDI (PDIred). The PDIred is then re-oxidized to PDIox by a coexisting cyclic diselenide compound, thereby restoring the function of PDI as a disulfide-forming agent. The produced diselenol state is readily oxidized to the original diselenide state with molecular oxygen, continuously sustaining the PDI catalytic cycle. This artificial electron relay system regulating enzymatic PDI function effectively promotes the oxidative folding of disulfide-containing proteins, such as insulin-a hypoglycemic formulation-by enhancing both yield and reaction velocity.

Keywords:  enzyme models * protein folding * redox chemistry * selenol * diselenide

DOI:  https://doi.org/10.1002/cbic.202400739
Nature. 2024 Nov 06.

A mechanism for hypoxia-induced inflammatory cell death in cancer.

Abhishek Bhardwaj, Maria C Panepinto, Beatrix Ueberheide, Benjamin G Neel.

Hypoxic cancer cells resist many antineoplastic therapies and can seed recurrence1,2. We previously found that either deficiency or inhibition of protein-tyrosine phosphatase (PTP1B) promotes human epidermal growth factor receptor 2-positive breast cancer cell death in hypoxia by activation of RNF213 (ref. 3), a large protein with multiple AAA-ATPase domains and two ubiquitin ligase domains (RING and RZ) implicated in Moyamoya disease, lipotoxicity and innate immunity4. Here we report that PTP1B and ABL1/2 reciprocally control RNF213 tyrosine phosphorylation and, consequently, its oligomerization and RZ domain activation. The RZ domain ubiquitylates and induces the degradation of the major NF-κB regulator CYLD/SPATA2. Decreased CYLD/SPATA2 levels lead to NF-κB activation and induction of the NLRP3 inflammasome which, together with hypoxia-induced endoplasmic reticulum stress, triggers pyroptotic cell death. Consistent with this model, CYLD deletion phenocopies, whereas NLRP3 deletion blocks, the effects of PTP1B deficiency on human epidermal growth factor receptor 2-positive breast cancer xenograft growth. Reconstitution studies with RNF213 mutants confirm that the RZ domain mediates tumour cell death. In concert, our results identify a unique, potentially targetable PTP1B-RNF213-CYLD-SPATA2 pathway critical for the control of inflammatory cell death in hypoxic tumours, provide new insights into RNF213 regulation and have potential implications for the pathogenesis of Moyamoya disease, inflammatory disorders and autoimmune disease.

DOI: https://doi.org/10.1038/s41586-024-08136-y
Adv Mater. 2024 Nov 03. e2412837

Augmenting Protein Degradation Capacity of PROTAC through Energy Metabolism Regulation and Targeted Drug Delivery.

Mixiao Tan, Xiaoyang Li, Long Cheng, Xianli Long, Guoliang Cao, Shengji Yu, Haitao Ran, Helin Feng, Hai Wang.

  The ubiquitin-proteasome system (UPS) is responsible for degrading over 70-80% of cellular proteins. Consequently, proteolysis-targeting chimeras (PROTACs) are developed to induce the ubiquitination and subsequent degradation of proteins of interest (POIs) by the UPS. To amplify the therapeutic efficacy of PROTACs, energy metabolism regulation is first harnessed to boost UPS function in tumor cells. Proteomic and ubiquitinome analyzes reveal that total ubiquitinated proteins and proteasome activity are significantly increased in 143B and MDA-MB-231 tumor cells following fasting-mimicking diet (FMD) treatment. As a result, the degradation efficiency of PROTACs targeting focal adhesion kinase (FAK-P) or bromodomain-containing protein 4 (BRD4-P) is significantly enhanced in FMD-treated 143B and MDA-MB-231 tumor cells. Then, silica-coated iron oxide nanoparticles are developed modified with tumor cell membranes for targeted delivery of PROTACs. Magnetic resonance imaging (MRI) and fluorescence imaging confirm that nanocarriers significantly improve the delivery efficiency of PROTACs in FMD-treated 143B or MDA-MB-231 tumors. In vivo studies demonstrate that the antitumor efficacy of FAK-P and BRD4-P is greatly augmented when combined with targeted delivery and FMD treatment. Overall, this study presents a strategy to enhance the efficacy of PROTACs in cancer therapy.

Keywords:  autophagy; energy metabolic reprogramming; fasting‐mimicking diet; proteolysis targeting chimera; ubiquitination

DOI:  https://doi.org/10.1002/adma.202412837
Cell. 2024 Oct 30. pii: S0092-8674(24)01188-7. [Epub ahead of print]

Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress.

Shiyun Cao, Sheena Faye Garcia, Huigang Shi, Ellie I James, Yuki Kito, Hui Shi, Haibin Mao, Sharon Kaisari, Gergely Rona, Sophia Deng, Hailey V Goldberg, Jackeline Ponce, Beatrix Ueberheide, Luca Lignitto, Miklos Guttman, Michele Pagano, Ning Zheng.

  Ubiquitin-dependent proteolysis regulates diverse cellular functions with high substrate specificity, which hinges on the ability of ubiquitin E3 ligases to decode the targets' degradation signals, i.e., degrons. Here, we show that BACH1, a transcription repressor of antioxidant response genes, features two distinct unconventional degrons encrypted in the quaternary structure of its homodimeric BTB domain. These two degrons are both functionalized by oxidative stress and are deciphered by two complementary E3s. FBXO22 recognizes a degron constructed by the BACH1 BTB domain dimer interface, which is unmasked from transcriptional co-repressors after oxidative stress releases BACH1 from chromatin. When this degron is impaired by oxidation, a second BACH1 degron manifested by its destabilized BTB dimer is probed by a pair of FBXL17 proteins that remodels the substrate into E3-bound monomers for ubiquitination. Our findings highlight the multidimensionality of protein degradation signals and the functional complementarity of different ubiquitin ligases targeting the same substrate.

Keywords:  BACH1; BTB; FBXL17; FBXO22; SCF; cullin-RING ligases; degron; dimer; heme; nitric oxide; oxidative stress; protein degradation; quaternary structure; transcription factor; ubiquitin ligase

DOI:  https://doi.org/10.1016/j.cell.2024.10.012
JCI Insight. 2024 Nov 05. pii: e175704. [Epub ahead of print]

LMAN1 serves as a cargo receptor for thrombopoietin.

Lesley A Everett, Zesen Lin, Ann Friedman, Vi T Tang, Greggory Myers, Ginette Balbin-Cuesta, Richard King, Guojing Zhu, Beth McGee, Rami Khoriaty.

  Thrombopoietin (TPO) is a plasma glycoprotein that binds its receptor on megakaryocytes (MK) and MK progenitors, resulting in enhanced platelet production. The mechanism by which TPO is secreted from hepatocytes remains poorly understood. LMAN1 and MCFD2 form a complex at the endoplasmic reticulum membrane, recruiting cargo proteins into COPII vesicles for secretion. In this study, we showed that LMAN1 deficient mice (with complete germline LMAN1 deficiency) exhibited mild thrombocytopenia, whereas the platelet count was entirely normal in mice with approximately 7% Lman1 expression. Surprisingly, mice deleted for Mcfd2 did not exhibit thrombocytopenia. Analysis of peripheral blood from LMAN1 deficient mice demonstrated normal platelet size and normal morphology of dense and alpha granules. LMAN1 deficient mice exhibited a trend toward reduced MK and MK progenitors in the bone marrow. We next showed that hepatocyte-specific but not hematopoietic Lman1 deletion results in thrombocytopenia, with plasma TPO level reduced in LMAN1 deficient mice, despite normal Tpo mRNA levels in LMAN1 deficient livers. TPO and LMAN1 interacted by co-immunoprecipitation in a heterologous cell line and TPO accumulated intracellularly in LMAN1 deleted cells. Altogether, these studies confirmed the hepatocyte as the cell of origin for TPO production in vivo and were consistent with LMAN1 as the endoplasmic reticulum cargo receptor that mediates the efficient secretion of TPO. To our knowledge, TPO is the first example of an LMAN1-dependent cargo that is independent of MCFD2.

Keywords:  Bone marrow; Hematology; Mouse models; Platelets

DOI:  https://doi.org/10.1172/jci.insight.175704
Proc Natl Acad Sci U S A. 2024 Nov 12. 121(46): e2404091121

Deciphering the RNA-binding protein network during endosomal mRNA transport.

Senthil-Kumar Devan, Sainath Shanmugasundaram, Kira Müntjes, Johannes Postma, Sander H J Smits, Florian Altegoer, Michael Feldbrügge.

  Microtubule-dependent endosomal transport is crucial for polar growth, ensuring the precise distribution of cellular cargos such as proteins and mRNAs. However, the molecular mechanism linking mRNAs to the endosomal surface remains poorly understood. Here, we present a structural analysis of the key RNA-binding protein Rrm4 from Ustilago maydis. Our findings reveal a different type of MademoiseLLE domain (MLLE) featuring a seven-helical bundle that provides a distinct binding interface. A comparative analysis with the canonical MademoiseLLE domain of the poly(A)-binding protein Pab1 disclosed unique characteristics of both domains. Deciphering the MLLE binding code enabled prediction and verification of previously unknown Rrm4 interactors containing short linear motifs. Importantly, we demonstrated that the human MLLE domains, such as those of PABPC1 and UBR5, employed a similar principle to distinguish among interaction partners. Thus, our study provides detailed mechanistic insights into how structural variations in the widely distributed MLLE domain facilitate mRNA attachment during endosomal transport.

Keywords:  PAM2; RNA transport; SLiM; Ustilago maydis; endosome

DOI:  https://doi.org/10.1073/pnas.2404091121
Proc Natl Acad Sci U S A. 2024 Nov 12. 121(46): e2412948121

An outcome-defining role for the triple-helical domain in regulating collagen-I assembly.

Kathryn M Yammine, Rasia C Li, Isabella M Borgula, Sophia Mirda Abularach, Andrew S DiChiara, Ronald T Raines, Matthew D Shoulders.

  Collagens are the foundational component of diverse tissues, including skin, bone, cartilage, and basement membranes, and are the most abundant protein class in animals. The fibrillar collagens are large, complex, multidomain proteins, all containing the characteristic triple helix motif. The most prevalent collagens are heterotrimeric, meaning that cells express at least two distinctive procollagen polypeptides that must assemble into specific heterotrimer compositions. The molecular mechanisms ensuring correct heterotrimeric assemblies are poorly understood - even for the most common collagen, type-I. The longstanding paradigm is that assembly is controlled entirely by the ~30 kDa globular C-propeptide (C-Pro) domain. Still, this dominating model for procollagen assembly has left many questions unanswered. Here, we show that the C-Pro paradigm is incomplete. In addition to the critical role of the C-Pro domain in templating assembly, we find that the amino acid sequence near the C terminus of procollagen's triple-helical domain plays an essential role in defining procollagen assembly outcomes. These sequences near the C terminus of the triple-helical domain encode conformationally stabilizing features that ensure only desirable C-Pro-mediated trimeric templates are committed to irreversible triple-helix folding. Incorrect C-Pro trimer assemblies avoid commitment to triple-helix formation thanks to destabilizing features in the amino acid sequences of their triple helix. Incorrect C-Pro assemblies are consequently able to dissociate and search for new binding partners. These findings provide a distinctive perspective on the mechanism of procollagen assembly, revealing the molecular basis by which incorrect homotrimer assemblies are avoided and setting the stage for a deeper understanding of the biogenesis of this ubiquitous protein.

Keywords:  endoplasmic reticulum; extracellular matrix; macromolecular complex; procollagen assembly; protein folding

DOI:  https://doi.org/10.1073/pnas.2412948121
J Am Chem Soc. 2024 Nov 05.

Discovery of CRBN-Dependent WEE1 Molecular Glue Degraders from a Multicomponent Combinatorial Library.

Hlib Razumkov, Zixuan Jiang, Kheewoong Baek, Inchul You, Qixiang Geng, Katherine A Donovan, Michelle T Tang, Rebecca J Metivier, Nada Mageed, Pooreum Seo, Zhengnian Li, Woong Sub Byun, Stephen M Hinshaw, Roman C Sarott, Eric S Fischer, Nathanael S Gray.

Small molecules promoting protein-protein interactions produce a range of therapeutic outcomes. Molecular glue degraders exemplify this concept due to their compact drug-like structures and ability to engage targets without reliance on existing cognate ligands. While cereblon molecular glue degraders containing glutarimide scaffolds have been approved for treatment of multiple myeloma and acute myeloid leukemia, the design of new therapeutically relevant monovalent degraders remains challenging. We report here an approach to glutarimide-containing molecular glue synthesis using multicomponent reactions as a central modular core-forming step. Screening the resulting library identified HRZ-1 derivatives that target casein kinase 1 α (CK1α) and Wee-like protein kinase (WEE1). Further medicinal chemistry efforts led to identification of selective monovalent WEE1 degraders that provide a potential starting point for the eventual development of a selective chemical degrader probe. The structure of the hit WEE1 degrader complex with CRBN-DDB1 and WEE1 provides a model of the protein-protein interface and ideas to rationalize the observed kinase selectivity. Our findings suggest that modular synthetic routes combined with in-depth structural characterization give access to selective molecular glue degraders and expansion of the CRBN-degradable proteome.

DOI: https://doi.org/10.1021/jacs.4c06127
Chem Biol Drug Des. 2024 Nov;104(5): e70009

CRBN-PROTACs in Cancer Therapy: From Mechanistic Insights to Clinical Applications.

Riya Thapa, Asif Ahmad Bhat, Gaurav Gupta, S Renuka Jyothi, Irwanjot Kaur, Sachin Kumar, Naveen Sharma, G V Siva Prasad, Atreyi Pramanik, Haider Ali.

  Cereblon (CRBN), a member of the E3 ubiquitin ligase complex, has gained significant attention as a therapeutic target in cancer. CRBN regulates the degradation of various proteins in cancer progression, including transcription factors and signaling molecules. PROTACs (proteolysis-targeting chimeras) are a novel approach that uses the cell's degradation system to remove disease-causing proteins selectively. CRBN-dependent PROTACs work by tagging harmful proteins for destruction through the ubiquitin-proteasome system. This strategy offers several advantages over traditional protein inhibition methods, including the potential to overcome drug resistance. Recent progress in developing CRBN-based PROTACs has shown promising preclinical results in both hematologic malignancies and solid tumors. Additionally, CRBN-based PROTACs have enhanced our understanding of CRBN's role in cancer, potentially serving as biomarkers for patient stratification and predicting therapeutic responses. In this review, we delineate the mechanisms of action for CRBN-dependent PROTACs (CRBN-PROTACs), summarize recent advances in preclinical and clinical applications, and provide our perspective on future development.

Keywords:  CRBN; Cereblon; E3 ubiquitin ligase; PROTACs; cancer therapy; drug resistance; solid tumors; targeted protein degradation; the ubiquitin–proteasome system

DOI:  https://doi.org/10.1111/cbdd.70009
Nat Cell Biol. 2024 Nov 01.

Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion.

Yue Lu, Tezin Walji, Benjamin Ravaux, Pratima Pandey, Changsong Yang, Bing Li, Delgermaa Luvsanjav, Kevin H Lam, Ruihui Zhang, Zhou Luo, Chuanli Zhou, Christa W Habela, Scott B Snapper, Rong Li, David J Goldhamer, David W Schmidtke, Duojia Pan, Tatyana M Svitkina, Elizabeth H Chen.

Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions is unclear. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two nucleation-promoting factors for the Arp2/3 complex, WAVE and N-WASP, exhibit different localization patterns in the protrusions. Whereas WAVE is closely associated with the plasma membrane at the leading edge of the protrusive structures, N-WASP is enriched with WIP along the actin bundles in the shafts of the protrusions. During protrusion initiation and growth, the Arp2/3 complex nucleates branched actin filaments to generate low-density actin clouds in which the large GTPase dynamin organizes the new branched actin filaments into bundles, followed by actin-bundle stabilization by WIP, the latter functioning as an actin-bundling protein. Disruption of any of these components results in defective protrusions and failed myoblast fusion in cultured cells and mouse embryos. Together, our study has revealed the intricate spatiotemporal coordination between two nucleation-promoting factors and two actin-bundling proteins in building invasive protrusions at the mammalian fusogenic synapse and has general implications in understanding invasive protrusion formation in cellular processes beyond cell-cell fusion.

DOI: https://doi.org/10.1038/s41556-024-01541-5
Sci Signal. 2024 Nov 05. 17(861): eadl6164

Extracellular proximal interaction profiling by cell surface-targeted TurboID reveals LDLR as a partner of liganded EGFR.

Rasha Al Mismar, Payman Samavarchi-Tehrani, Brendon Seale, Vesal Kasmaeifar, Claire E Martin, Anne-Claude Gingras.

Plasma membrane proteins play pivotal roles in receiving and transducing signals from other cells and from the environment and are vital for cellular functionality. Enzyme-based, proximity-dependent approaches, such as biotin identification (BioID), combined with mass spectrometry have begun to illuminate the landscape of proximal protein interactions within intracellular compartments. To extend the potential of these approaches to study the extracellular environment, we developed extracellular TurboID (ecTurboID), a method designed to profile the interactions between proteins on the surfaces of living cells over short timescales using the fast-acting biotin ligase TurboID. After optimizing our experimental and data analysis strategies to capture extracellular proximity interactions, we used ecTurboID to reveal the proximal interactomes of several plasma membrane proteins, including the epidermal growth factor receptor (EGFR). We found that EGF stimulation induced an association between EGFR and the low-density lipoprotein receptor (LDLR) and changed the interactome of LDLR by increasing its proximity with proteins that regulate EGFR signaling. The identification of this interaction between two well-studied and clinically relevant receptors illustrates the utility of our modified proximity labeling methodology for identifying dynamic extracellular associations between plasma membrane proteins.

DOI: https://doi.org/10.1126/scisignal.adl6164
STAR Protoc. 2024 Nov 02. pii: S2666-1667(24)00451-9. [Epub ahead of print]5(4): 103286

Protocol for mapping differential protein-protein interaction networks using affinity purification-mass spectrometry.

Prashant Kaushal, Manisha R Ummadi, Gwendolyn M Jang, Yennifer Delgado, Sara K Makanani, Kareem Alba, Declan M Winters, Sophie F Blanc, Jiewei Xu, Benjamin Polacco, Yuan Zhou, Erica Stevenson, Manon Eckhardt, Lorena Zuliani-Alvarez, Robyn Kaake, Danielle L Swaney, Nevan J Krogan, Mehdi Bouhaddou.

  Proteins congregate into complexes to perform diverse cellular functions. Protein complexes are remodeled by protein-coding mutations or cellular signaling changes, driving phenotypic outcomes in health and disease. We present an affinity purification-mass spectrometry (AP-MS) proteomics protocol to express affinity-tagged "bait" proteins in mammalian cells, identify and quantify purified protein interactors, and visualize differential protein-protein interaction networks between pairwise conditions. Our protocol possesses general applicability to various cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al.1.

Keywords:  Mass Spectrometry; Molecular Biology; Protein Biochemistry; Protein expression and purification; Proteomics; Systems biology

DOI:  https://doi.org/10.1016/j.xpro.2024.103286
Cell Death Discov. 2024 Nov 01. 10(1): 462

Targeting the HECTD3-p62 axis increases the radiosensitivity of triple negative breast cancer cells.

Maobo Huang, Wenjing Liu, Zhuo Cheng, Fubing Li, Yanjie Kong, Chuanyu Yang, Yu Tang, Dewei Jiang, Wenhui Li, Yudie Hu, Jinhui Hu, PemaTenzin Puno, Ceshi Chen.

Triple negative breast cancer is the most malignant subtype of breast cancer and current treatment options are limited. Radiotherapy is one of the primary therapeutic options for patients with TNBC. In this study, we discovered that the E3 ubiquitin ligase, HECTD3, promoted TNBC cell survival after irradiation. HECTD3 collaborated with UbcH5b to promote p62 ubiquitination and autophagy while HECTD3 deletion led to p62 accumulation in the nucleus in response to irradiation, thus inhibiting RNF168 mediated DNA damage repair. Furthermore, the HECTD3/UbcH5b inhibitor, PC3-15, increased the radiosensitivity of TNBC cells by inhibiting DNA damage repair. Taken together, we conclude that HECTD3 promotes autophagy and DNA damage repair in response to irradiation in a p62-denpendent manner, and that inhibition of the HECTD3-p62 axis could be a potential therapeutic strategy for patients with TNBC in addition to radiotherapy.

DOI: https://doi.org/10.1038/s41420-024-02154-5
Sci Adv. 2024 Nov 08. 10(45): eadq0660

Endosomes serve as signaling platforms for RIG-I ubiquitination and activation.

Kuan-Ru Chen, Chia-Yu Yang, San-Ging Shu, Yin-Chiu Lo, Kuan-Wei Lee, Li-Chun Wang, Jia-Bao Chen, Meng-Cen Shih, Hung-Chun Chang, Yu-Ju Hsiao, Chao-Liang Wu, Tse-Hua Tan, Pin Ling.

RIG-I-like receptors (RLRs) are cytosolic RNA sensors critical for antiviral immunity. RLR activation is regulated by polyubiquitination and oligomerization following RNA binding. Yet, little is known about how RLRs exploit subcellular organelles to facilitate their posttranslational modifications and activation. Endosomal adaptor TAPE regulates the endosomal TLR and cytosolic RLR pathways. The potential interplay between RIG-I signaling and endosomes has been explored. Here, we report that endosomes act as platforms for facilitating RIG-I polyubiquitination and complex formation. RIG-I was translocated onto endosomes to form signaling complexes upon activation. Ablation of endosomes impaired RIG-I signaling to type I IFN activation. TAPE mediates the interaction and polyubiquitination of RIG-I and TRIM25. TAPE-deficient myeloid cells were defective in type I IFN activation upon RNA ligand and virus challenges. Myeloid TAPE deficiency increased the susceptibility to RNA virus infection in vivo. Our work reveals endosomes as signaling platforms for RIG-I activation and antiviral immunity.

DOI: https://doi.org/10.1126/sciadv.adq0660
J Mol Biol. 2024 Oct 26. pii: S0022-2836(24)00469-8. [Epub ahead of print] 168840

The HSP90/R2TP quaternary chaperone scaffolds assembly of the TSC complex.

Claire Abéza, Philipp Busse, Ana C F Paiva, Marie-Eve Chagot, Justine Schneider, Marie-Cécile Robert, Franck Vandermoere, Christine Schaeffer, Bruno Charpentier, Pedro M F Sousa, Tiago M Bandeiras, Xavier Manival, Sarah Cianferani, Edouard Bertrand, Céline Verheggen.

  The R2TP chaperone is composed of the RUVBL1/RUVBL2 AAA+ ATPases and two adapter proteins, RPAP3 and PIH1D1. Together with HSP90, it functions in the assembly of macromolecular complexes that are often involved in cell proliferation. Here, proteomic experiments using the isolated PIH domain reveals additional R2TP partners, including the Tuberous Sclerosis Complex (TSC) and many transcriptional complexes. The TSC is a key regulator of mTORC1 and is composed of TSC1, TSC2 and TBC1D7. We show a direct interaction of TSC1 with the PIH phospho-binding domain of PIH1D1, which is, surprisingly, phosphorylation independent. Via the use of mutants and KO cell lines, we observe that TSC2 makes independent interactions with HSP90 and the TPR domains of RPAP3. Moreover, inactivation of PIH1D1 or the RUVBL1/2 ATPase activity inhibits the association of TSC1 with TSC2. Taken together, these data suggest a model in which the R2TP recruits TSC1 via PIH1D1 and TSC2 via RPAP3 and HSP90, and use the chaperone-like activities of RUVBL1/2 to stimulate their assembly.

Keywords:  2); ATPases associated with diverse cellular activities (RUVBL1; R2TP; Tuberous sclerosis complex (TSC); heat shock protein 90 (HSP90); protein‐protein interaction

DOI:  https://doi.org/10.1016/j.jmb.2024.168840
J Biol Chem. 2024 Nov 02. pii: S0021-9258(24)02456-6. [Epub ahead of print] 107954

The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay.

Wen Zheng, Jinjing Guo, Shuyan Ma, Rong Sun, Yihua Song, Yuanmeng Chen, Renfang Mao, Yihui Fan.

  3'-Untranslated regions (3'UTRs) are recognized for their role in regulating mRNA turnover while the turnover of a specific group of mRNAs mediated by coding sequences (CDS) remains poorly understood. N4BP1 is a critical inflammatory regulator in vivo with a molecular mechanism that is not yet clearly defined. Our study reveals that N4BP1 efficiently degrades its mRNA targets via CDS rather than the 3'-UTR. This CDS-dependent mRNA turnover mechanism appears to be a general feature of N4BP1, as evidenced by testing multiple mRNA substrates, such as Fos-C, Fos-B, Jun-B and CXCL1. Detailed mapping of the motif identified a crucial 33nt (289-322) sequence near the 5'-end of Fos-C-CDS, where the presence of polyC is necessary for N4BP1-mediated degradation. Functional studies involving domain deletion and point mutations showed that both the KH and NYN domains are essential for N4BP1 to restrict mRNA substrates. The function of N4BP1 in mRNA turnover is not dependent on nonsense-mediated decay as it efficiently restricts mRNA substrates even in cells deficient in UPF1, UPF3A, and UPF3B. Additionally, the function of N4BP1 is not reliant on LUC7L3 despite its known association with this protein. Our findings suggest that N4BP1 acts as an endoribonuclease to degrade mRNA substrates primarily through coding sequences containing a C-rich motif.

Keywords:  C-rich motif; Fos-C; KH domain; N4BP1; NYN domain; nonsense-mediated mRNA decay

DOI:  https://doi.org/10.1016/j.jbc.2024.107954