bims-proteo 2023-10-15 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–10–15
29 papers selected by
Eric Chevet, INSERM

Mechanistic insights into the enzymatic activity of E3 ligase HOIL-1L and its regulation by the linear ubiquitin chain binding.
Multiple quality control mechanisms monitor yeast chitin synthase folding in the endoplasmic reticulum.
Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2.
Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis.
The cryo-EM structure of the human ERAD retrotranslocation complex.
Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.
Native mass spectrometry interrogation of complexes formed during targeted protein degradation.
A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion.
ATNC: Versatile Nanobody Chimeras for Autophagic Degradation of Intracellular Unligandable and Undruggable Proteins.
Tetracyclines activate mitoribosome quality control and reduce ER stress to promote cell survival.
Vault-phagy: a phase-separation-mediated selective autophagy of vault, a non-membranous organelle.
Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5.
Interference with the HNF4-dependent gene regulatory network diminishes endoplasmic reticulum stress in hepatocytes.
The deubiquitinase ZRANB1 is an E3 ubiquitin ligase for SLC7A11 and regulates ferroptotic resistance.
TRIM55 promotes noncanonical NF-κB signaling and B cell-mediated immune responses by coordinating p100 ubiquitination and processing.
The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.
C9orf72-catalyzed GTP loading of Rab39A enables HOPS-mediated membrane tethering and fusion in mammalian autophagy.
Identification of a Novel Hypoxia-induced Inflammatory Cell Death Pathway.
Targeted Protein Degradation Mediated by Genetically Engineered Lysosome-Targeting Exosomes.
AMPK Regulates Phagophore-to-Autophagosome Maturation.
SUMO in the regulation of DNA repair and transcription at nuclear pores.
Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control.
Activation of human STING by a molecular glue-like compound.
A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint.
Proteogenetic drug response profiling elucidates targetable vulnerabilities of myelofibrosis.
Higher-order structural organization of the mitochondrial proteome charted by in situ cross-linking mass spectrometry.
Apoptotic stress causes mtDNA release during senescence and drives the SASP.
Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility.
Lipid droplets modulate proteostasis, SQST-1/SQSTM1 dynamics, and lifespan in C. elegans.

Sci Adv. 2023 Oct 13. 9(41): eadi4599

Mechanistic insights into the enzymatic activity of E3 ligase HOIL-1L and its regulation by the linear ubiquitin chain binding.

Xiaolong Xu, Yaru Wang, Yan Zhang, Yingli Wang, Yue Yin, Chao Peng, Xinyu Gong, Miao Li, Yuchao Zhang, Mingfang Zhang, Yubin Tang, Xindi Zhou, Haobo Liu, Lifeng Pan.

Heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) serves as a unique E3 ligase to catalyze the mono-ubiquitination of relevant protein or sugar substrates and plays vital roles in numerous cellular processes in mammals. However, the molecular mechanism underpinning the E3 activity of HOIL-1L and the related regulatory mechanism remain elusive. Here, we report the crystal structure of the catalytic core region of HOIL-1L and unveil the key catalytic triad residues of HOIL-1L. Moreover, we discover that HOIL-1L contains two distinct linear di-ubiquitin binding sites that can synergistically bind to linear tetra-ubiquitin, and the binding of HOIL-1L with linear tetra-ubiquitin can promote its E3 activity. The determined HOIL-1L/linear tetra-ubiquitin complex structure not only elucidates the detailed binding mechanism of HOIL-1L with linear tetra-ubiquitin but also uncovers a unique allosteric ubiquitin-binding site for the activation of HOIL-1L. In all, our findings provide mechanistic insights into the E3 activity of HOIL-1L and its regulation by the linear ubiquitin chain binding.

DOI: https://doi.org/10.1126/sciadv.adi4599
Mol Biol Cell. 2023 Oct 11. mbcE23050186

Multiple quality control mechanisms monitor yeast chitin synthase folding in the endoplasmic reticulum.

Noelia Sanchez, Nagore de Leon, Rosario Valle, Jia Jun Fung, Anton Khmelinskii, Cesar Roncero.

The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD). Here we show that proper N-glycosylation of its luminal domain is essential to prevent the aggregation of the protein and its subsequent recognition by the Hrd1-dependent ERAD-L machinery. In addition, the interaction of Chs3 with its chaperone Chs7 seems to mask additional cytosolic degrons, thereby avoiding their recognition by the ERAD-C pathway. On top of that, Chs3 molecules that are not degraded by conventional ERAD can move along the ER membrane to reach the inner nuclear membrane, where they are degraded by the inner nuclear membrane-associated degradation (INMAD) system, which contributes to the intracellular homeostasis of Chs3. These results indicate that Chs3 is an excellent model to study quality control mechanisms in the cell and reinforce its role as a paradigm in intracellular trafficking research.

DOI: https://doi.org/10.1091/mbc.E23-05-0186
Nat Commun. 2023 Oct 10. 14(1): 6345

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2.

Sarath Ramachandran, Nikolai Makukhin, Kevin Haubrich, Manjula Nagala, Beth Forrester, Dylan M Lynch, Ryan Casement, Andrea Testa, Elvira Bruno, Rosaria Gitto, Alessio Ciulli.

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

DOI: https://doi.org/10.1038/s41467-023-41894-3
Mol Cell. 2023 Oct 08. pii: S1097-2765(23)00753-0. [Epub ahead of print]

Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis.

Yi Fu, Olivia Sacco, Emily DeBitetto, Evgeny Kanshin, Beatrix Ueberheide, Agnel Sfeir.

  Mitochondrial DNA double-strand breaks (mtDSBs) lead to the degradation of circular genomes and a reduction in copy number; yet, the cellular response in human cells remains elusive. Here, using mitochondrial-targeted restriction enzymes, we show that a subset of cells with mtDSBs exhibited defective mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy confirmed the altered mitochondrial membrane and cristae ultrastructure. Intriguingly, mtDSBs triggered the integrated stress response (ISR) via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by DELE1 and heme-regulated eIF2α kinase (HRI). When ISR was inhibited, the cells experienced intensified mitochondrial defects and slower mtDNA recovery post-breakage. Lastly, through proteomics, we identified ATAD3A-a membrane-bound protein interacting with nucleoids-as potentially pivotal in relaying signals from impaired genomes to the inner mitochondrial membrane. In summary, our study delineates the cascade connecting damaged mitochondrial genomes to the cytoplasm and highlights the significance of the ISR in maintaining mitochondrial homeostasis amid genome instability.

Keywords:  ATAD3A; double-strand breaks; integrated stress response; mitochondrial DNA; protein import

DOI:  https://doi.org/10.1016/j.molcel.2023.09.026
Sci Adv. 2023 Oct 13. 9(41): eadi5656

The cryo-EM structure of the human ERAD retrotranslocation complex.

Bing Rao, Qian Wang, Deqiang Yao, Ying Xia, Wenguo Li, Yuming Xie, Shaobai Li, Mi Cao, Yafeng Shen, An Qin, Jie Zhao, Yu Cao.

Endoplasmic reticulum-associated degradation (ERAD) maintains protein homeostasis by retrieving misfolded proteins from the endoplasmic reticulum (ER) lumen into the cytosol for degradation. The retrotranslocation of misfolded proteins across the ER membrane is an energy-consuming process, with the detailed transportation mechanism still needing clarification. We determined the cryo-EM structures of the hetero-decameric complex formed by the Derlin-1 tetramer and the p97 hexamer. It showed an intriguing asymmetric complex and a putative coordinated squeezing movement in Derlin-1 and p97 parts. With the conformational changes of p97 induced by its ATP hydrolysis activities, the Derlin-1 channel could be torn into a "U" shape with a large opening to the lipidic environment, thereby forming an entry for the substrates in the ER membrane. The EM analysis showed that p97 formed a functional protein complex with Derlin-1, revealing the coupling mechanism between the ERAD retrotranslocation and the ATP hydrolysis activities.

DOI: https://doi.org/10.1126/sciadv.adi5656
Dev Cell. 2023 10 09. pii: S1534-5807(23)00445-8. [Epub ahead of print]58(19): 1950-1966.e8

Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

Nari Kim, Tae-Hyeon Kim, Chaelim Kim, Jee-Eun Lee, Myeong-Gyun Kang, Sanghee Shin, Minkyo Jung, Jong-Seo Kim, Ji Young Mun, Hyun-Woo Rhee, Seung-Yeol Park, Yongdae Shin, Joo-Yeon Yoo.

  Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.

Keywords:  COPII; ER-to-Golgi; IDP; IDR; SCOTIN; SHISA-5; bimolecular condensates; endoplasmic reticulum; interferon; intrinsically disordered region

DOI:  https://doi.org/10.1016/j.devcel.2023.08.030
Rapid Commun Mass Spectrom. 2023 Nov 30. 37(22): e9604

Native mass spectrometry interrogation of complexes formed during targeted protein degradation.

Ikhlas M M Ahmed, Rebecca Beveridge.

RATIONALE: Protein degraders are small molecules that promote cellular degradation of a target protein. Degraders simultaneously bind to their target and an E3 ligase, bringing them into close spatial proximity, but the formation of this ternary complex is difficult to measure with many biophysical techniques.
METHODS: Native mass spectrometry (nMS) is an effective label-free technique to identify the complexes formed by proteolysis-targeting chimeras (PROTACs). It can monitor the formation of ternary E3-PROTAC-target complexes and detect intermediate binary species. Experiments are described using a Synapt G2Si (Waters) equipped with a nano-electrospray ionisation source.
RESULTS: The protocol describes nMS experiments for measuring the complexes formed by PROTAC molecules. It also describes how to investigate differences in the affinity of PROTAC complexes, whether a PROTAC shows specificity for a given target and whether a PROTAC shows cooperative behaviour.
CONCLUSIONS: Here, we provide step-by-step instructions for the sample preparation of PROTAC complexes and their nMS interrogation to obtain optimal information on their binding modes.

DOI: https://doi.org/10.1002/rcm.9604
Sci Adv. 2023 Oct 13. 9(41): eadh1134

A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion.

Jie Li, Gregory J Krause, Qi Gui, Susmita Kaushik, Gergely Rona, Qingyue Zhang, Feng-Xia Liang, Avantika Dhabaria, Carlos Anerillas, Jennifer L Martindale, Nikita Vasilyev, Manor Askenazi, Beatrix Ueberheide, Evgeny Nudler, Myriam Gorospe, Ana Maria Cuervo, Michele Pagano.

Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.

DOI: https://doi.org/10.1126/sciadv.adh1134
J Am Chem Soc. 2023 Oct 12.

ATNC: Versatile Nanobody Chimeras for Autophagic Degradation of Intracellular Unligandable and Undruggable Proteins.

Huiping He, Chengjian Zhou, Xi Chen.

Targeted protein degradation (TPD) through the autophagy pathway displays broad substrate scope and is gaining increasing interest in biology and medicine. However, current approaches using small-molecule degraders have limitations due to the lack of versatility, modularity, and ease of implementation and are restricted to addressing only ligandable proteins. Herein, we report a nonsmall molecule-based autophagy-targeting nanobody chimera (ATNC), or phagobody, for selective degradation of intracellular targets, which overcomes these limitations. The core of an ATNC features a nanobody for recruiting proteins as well as an autophagic pathway-directing module. ATNC turns out to be a general, modular, and versatile degradation platform. We show that ATNC can be versatilely implemented in different ways including expressed ATNC intrabodies for ease of use, chemically induced proximity (CIP)-operated logic-gated conditional and tunable degradation, and cyclic cell-penetrating peptide-tethered cell-permeable phagobodies that selectively degrade the undruggable therapeutically relevant HE4 protein, resulting in effective suppression of ovarian cancer cell proliferation and migration. Overall, ATNC represents a general, modular, and versatile targeted degradation platform that degrades unligandable proteins and offers therapeutic potential.

DOI: https://doi.org/10.1021/jacs.3c08843
EMBO Rep. 2023 Oct 11. e57228

Tetracyclines activate mitoribosome quality control and reduce ER stress to promote cell survival.

Conor T Ronayne, Thomas D Jackson, Christopher F Bennett, Elizabeth A Perry, Noa Kantorovic, Pere Puigserver.

  Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations is redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. One method to rescue this cell death vulnerability is the inhibition of mitochondrial translation using tetracyclines. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that tetracyclines inhibit the mitochondrial ribosome and promote survival through suppression of endoplasmic reticulum (ER) stress. Tetracyclines increase mitochondrial levels of the mitoribosome quality control factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) and promote its recruitment to the mitoribosome large subunit, where MALSU1 is necessary for tetracycline-induced survival and suppression of ER stress. Glucose starvation induces ER stress to activate the unfolded protein response and IRE1α-mediated cell death that is inhibited by tetracyclines. These studies establish a new interorganelle communication whereby inhibition of the mitoribosome signals to the ER to promote survival, implicating basic mechanisms of cell survival and treatment of mitochondrial diseases.

Keywords:  IRE1α; MALSU1; mitochondrial disease; mitoribosome; tetracyclines

DOI:  https://doi.org/10.15252/embr.202357228
Autophagy. 2023 Oct 10. 1-2

Vault-phagy: a phase-separation-mediated selective autophagy of vault, a non-membranous organelle.

Reo Kurusu, Hideaki Morishita, Masaaki Komatsu.

  SQSTM1/p62 bodies are phase-separated condensates that play a fundamental role in intracellular quality control and stress responses. Despite extensive studies investigating the mechanism of formation and degradation of SQSTM1/p62 bodies, the constituents of SQSTM1/p62 bodies remain elusive. We recently developed a purification method for intracellular SQSTM1/p62 bodies using a cell sorter and identified their constituents by mass spectrometry. Combined with mass spectrometry of tissues from selective autophagy-deficient mice, we identified vault, a ubiquitous non-membranous organelle composed of proteins and non-coding RNA, as a novel substrate for selective autophagy. Vault directly binds to NBR1, an SQSTM1/p62 binding partner recruited to SQSTM1/p62 bodies, and is subsequently degraded by selective autophagy dependent on the phase separation of SQSTM1/p62. We named this process "vault-phagy" and found that defects in vault-phagy are related to nonalcoholic steatohepatitis (NASH)-derived hepatocellular carcinoma. Our method for purifying SQSTM1/p62 bodies will contribute to elucidating the mechanisms of several stress responses and diseases mediated by SQSTM1/p62 bodies.

Keywords:  Fluorescence-activated particle sorting; Mallory-Denk bodies; NBR1; liquid-liquid phase separation; nonalcoholic steatohepatitis; selective autophagy

DOI:  https://doi.org/10.1080/15548627.2023.2266996
Brain. 2023 Oct 07. pii: awad340. [Epub ahead of print]

Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5.

Camilla Aurora Franchino, Martina Brughera, Valentina Baderna, Daniele De Ritis, Alessandra Rocco, Sara Seneca, Luc Regal, Paola Podini, Maurizio D'Antonio, Camilo Toro, Angelo Quattrini, Emmanuel Scalais, Francesca Maltecca.

  AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane (IMM). Heterozygous AFG3L2 mutations cause Spinocerebellar Ataxia type 28 (SCA28) or Dominant Optic Atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe Spastic Ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia, and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the over-activation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the IMM protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response (ISR). In general, the ISR reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented so far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the ISR. We indeed found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of ISR via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts, and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons (PNs). Notably, Sephin-1 treatment in vivo extended the life span of Afg3l2-/- mice, improved PN morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the ISR may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.

Keywords:  OMA1; integrated stress response; spastic ataxia type 5

DOI:  https://doi.org/10.1093/brain/awad340
Hepatol Commun. 2023 11 01. pii: e0278. [Epub ahead of print]7(11):

Interference with the HNF4-dependent gene regulatory network diminishes endoplasmic reticulum stress in hepatocytes.

Anit Shah, Ian Huck, Kaylia Duncan, Erica R Gansemer, Kaihua Liu, Reed C Adajar, Udayan Apte, Mark A Stamnes, D Thomas Rutkowski.

BACKGROUND: In all eukaryotic cell types, the unfolded protein response (UPR) upregulates factors that promote protein folding and misfolded protein clearance to help alleviate endoplasmic reticulum (ER) stress. Yet, ER stress in the liver is uniquely accompanied by the suppression of metabolic genes, the coordination and purpose of which are largely unknown.
METHODS: Here, we combined in silico machine learning, in vivo liver-specific deletion of the master regulator of hepatocyte differentiation HNF4α, and in vitro manipulation of hepatocyte differentiation state to determine how the UPR regulates hepatocyte identity and toward what end.
RESULTS: Machine learning identified a cluster of correlated genes that were profoundly suppressed by persistent ER stress in the liver. These genes, which encode diverse functions including metabolism, coagulation, drug detoxification, and bile synthesis, are likely targets of the master regulator of hepatocyte differentiation HNF4α. The response of these genes to ER stress was phenocopied by liver-specific deletion of HNF4α. Strikingly, while deletion of HNF4α exacerbated liver injury in response to an ER stress challenge, it also diminished UPR activation and partially preserved ER ultrastructure, suggesting attenuated ER stress. Conversely, pharmacological maintenance of hepatocyte identity in vitro enhanced sensitivity to stress.
CONCLUSIONS: Together, our findings suggest that the UPR regulates hepatocyte identity through HNF4α to protect ER homeostasis even at the expense of liver function.

DOI: https://doi.org/10.1097/HC9.0000000000000278
J Cell Biol. 2023 Nov 06. pii: e202212072. [Epub ahead of print]222(11):

The deubiquitinase ZRANB1 is an E3 ubiquitin ligase for SLC7A11 and regulates ferroptotic resistance.

Shan Huang, Qimin Zhang, Manyu Zhao, Xing Wang, Yilei Zhang, Boyi Gan, Peijing Zhang.

The dependency of cancer cells on iron increases their susceptibility to ferroptosis, thus providing new opportunities for patients with treatment-resistant tumors. However, we show that lipid peroxidation, a hallmark of ferroptosis, was found in various areas of patient samples, indicating the potential resistance of ferroptosis. Using whole deubiquitinases (DUBs) sgRNA screening, we found that loss of ZRANB1 confers cancer cell resistance to ferroptosis. Intriguingly, functional studies revealed that ZRANB1 ubiquitinates and represses SLC7A11 expression as an E3 ubiquitin ligase and that ZRANB1 inhibits glutathione (GSH) synthesis through SLC7A11 degradation, leading to elevated lipid peroxidation and ferroptosis. Deletion of the region (residues 463-584) abolishes the E3 activity of ZRANB1. Moreover, we show that ZRANB1 has lower expression in tumors, which is positively correlated with lipid peroxidation. Collectively, our results demonstrate the role of ZRANB1 in ferroptosis resistance and unveil mechanisms involving modulation of E3 ligase activity through an unconventional catalytic domain.

DOI: https://doi.org/10.1083/jcb.202212072
Sci Signal. 2023 Oct 10. 16(806): eabn5410

TRIM55 promotes noncanonical NF-κB signaling and B cell-mediated immune responses by coordinating p100 ubiquitination and processing.

Liangbin Lin, Hui Yu, Li Li, Wenyong Yang, Xueying Chen, Yanqiu Gong, Qingqiang Lei, Zhonghan Li, Zhaocai Zhou, Lunzhi Dai, Huiyuan Zhang, Hongbo Hu.

The ubiquitination-dependent processing of NF-κB2 (also known as p100) is a critical step in the activation of the noncanonical NF-κB pathway. We investigated the molecular mechanisms regulating this process and showed that TRIM55 was the E3 ubiquitin ligase that mediated the ubiquitination of p100 and coordinated its processing. TRIM55 deficiency impaired noncanonical NF-κB activation and B cell function. Mice with a B cell-specific Trim55 deficiency exhibited reduced germinal center formation and antibody production. These mice showed less severe symptoms than those of control mice upon the induction of a systemic lupus-like disease, suggesting B cell-intrinsic functions of TRIM55 in humoral immune responses and autoimmunity. Mechanistically, the ubiquitination of p100 mediated by TRIM55 was crucial for p100 processing by VCP, an ATPase that mediates ubiquitin-dependent protein degradation by the proteasome. Furthermore, we found that TRIM55 facilitated the interaction between TRIM21 and VCP as well as TRIM21-mediated K63-ubiquitination of VCP, both of which were indispensable for the formation of the VCP-UFD1-NPL4 complex and p100 processing. Together, our results reveal a mechanism by which TRIM55 fine-tunes p100 processing and regulates B cell-dependent immune responses in vivo, highlighting TRIM55 as a potential therapeutic target for lupus-like disease.

DOI: https://doi.org/10.1126/scisignal.abn5410
Cell Rep. 2023 Oct 09. pii: S2211-1247(23)01241-X. [Epub ahead of print]42(10): 113229

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Vidhyadhar Nandana, Imalka W Rathnayaka-Mudiyanselage, Nisansala S Muthunayake, Ali Hatami, C Bruce Mousseau, Luis A Ortiz-Rodríguez, Jamuna Vaishnav, Michael Collins, Alisa Gega, Kaveendya S Mallikaarachchi, Hadi Yassine, Aishwarya Ghosh, Julie S Biteen, Yingxi Zhu, Matthew M Champion, W Seth Childers, Jared M Schrader.

  Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

Keywords:  BR-bodies; CP: Cell biology; CP: Molecular biology; RNA degradosome; RNase E; biomolecular condensates; mRNA decay

DOI:  https://doi.org/10.1016/j.celrep.2023.113229
Nat Commun. 2023 Oct 11. 14(1): 6360

C9orf72-catalyzed GTP loading of Rab39A enables HOPS-mediated membrane tethering and fusion in mammalian autophagy.

Shen Zhang, Mindan Tong, Denghao Zheng, Huiying Huang, Linsen Li, Christian Ungermann, Yi Pan, Hanyan Luo, Ming Lei, Zaiming Tang, Wan Fu, She Chen, Xiaoxia Liu, Qing Zhong.

The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet reconstituting the mammalian HOPS complex remains a challenge. Here we propose a "hook-up" model for mammalian HOPS complex assembly, which requires two HOPS sub-complexes docking on membranes via membrane-associated Rabs. We identify Rab39A as a key small GTPase that recruits HOPS onto autophagic vesicles. Proper pairing with Rab2 and Rab39A enables HOPS complex assembly between proteoliposomes for its tethering function, facilitating efficient membrane fusion. GTP loading of Rab39A is important for the recruitment of HOPS to autophagic membranes. Activation of Rab39A is catalyzed by C9orf72, a guanine exchange factor associated with amyotrophic lateral sclerosis and familial frontotemporal dementia. Constitutive activation of Rab39A can rescue autophagy defects caused by C9orf72 depletion. These results therefore reveal a crucial role for the C9orf72-Rab39A-HOPS axis in autophagosome-lysosome fusion.

DOI: https://doi.org/10.1038/s41467-023-42003-0
bioRxiv. 2023 Sep 26. pii: 2023.08.05.552118. [Epub ahead of print]

Identification of a Novel Hypoxia-induced Inflammatory Cell Death Pathway.

Abhishek Bhardwaj, Maria Antonelli, Beatrix Ueberheide, Benjamin G Neel.

Hypoxic cancer cells resist many anti-neoplastic therapies and can seed recurrence. We found previously that PTP1B deficiency promotes HER2+ breast cancer cell death in hypoxia by activating RNF213, an ∼600kDa protein containing AAA-ATPase domains and two ubiquitin ligase domains (RING and RZ) that also is implicated in Moyamoya disease (MMD), lipotoxicity, and innate immunity. Here we report that PTP1B and ABL1/2 reciprocally control RNF213 phosphorylation on tyrosine-1275. This phosphorylation promotes RNF213 oligomerization and RZ domain activation. The RZ domain ubiquitylates CYLD/SPATA2, and together with the LUBAC complex, induces their degradation. Decreased CYLD/SPATA2 causes NF-κB activation, which together with hypoxia-induced ER-stress triggers GDSMD-dependent pyroptosis. Mutagenesis experiments show that the RING domain negatively regulates the RZ domain. CYLD -deleted HER2+ cell-derived xenografts phenocopy the effects of PTP1B deficiency, and reconstituting RNF213 knockout lines with RNF213 mutants shows that the RZ domain mediates PTP1B-dependent tumor cell death. Our results identify a novel, potentially targetable PTP1B/RNF213/CYCLD/SPATA pathway critical for controlling inflammatory cell death in hypoxic tumors that could be exploited to target hypoxic tumor cells, potentially turning "cold" tumors "hot". Our findings also reveal new insights into RNF213 regulation, and have potentially important implications for the pathogenesis of MMD, atherosclerosis, and inflammatory and auto-immune disorders.

DOI: https://doi.org/10.1101/2023.08.05.552118
Nano Lett. 2023 Oct 12.

Targeted Protein Degradation Mediated by Genetically Engineered Lysosome-Targeting Exosomes.

Tao Wang, Liang Sun, Tianyu Ren, Min Hou, Ying Long, Jian-Hui Jiang, Jianjun He.

  Protein-degrading chimeras are superior drug modalities compared to traditional protein inhibitors because of their effective therapeutic performance. So far, various targeted protein degradation strategies, including proteolysis-targeting chimeras and lysosome-targeting chimeras, have emerged as essential technologies for tackling diseases caused by abnormal protein expression. Here, we report the development and application of lysosome-targeting exosomes (LYTEXs) for the selective degradation of membrane protein targets. LYTEXs are genetically engineered exosomes expressing multivalent single-chain fragment variables, simultaneously recognizing cell-surface lysosome-targeting and to-be-degraded protein. We show that by targeting the lysosome-directing asialoglycoprotein receptor, bispecific LYTEXs can induce lysosomal degradation of membrane-associated therapeutic targets. This strategy provides a generalizable, easy-to-prepare platform for modulating surface protein expression, with the advantage of therapeutic delivery.

Keywords:  ASGPR; exosome; lysosomal degradation; targeted protein degradation

DOI:  https://doi.org/10.1021/acs.nanolett.3c03148
bioRxiv. 2023 Sep 30. pii: 2023.09.28.559981. [Epub ahead of print]

AMPK Regulates Phagophore-to-Autophagosome Maturation.

Carlo Barnaba, David G Broadbent, Gloria I Perez, Jens C Schmidt.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPK's regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

DOI: https://doi.org/10.1101/2023.09.28.559981
FEBS Lett. 2023 Oct 07.

SUMO in the regulation of DNA repair and transcription at nuclear pores.

Susan M Gasser, Françoise Stutz.

  Two related post-translational modifications, the covalent linkage of Ubiquitin and the Small Ubiquitin-related MOdifier (SUMO) to lysine residues, play key roles in the regulation of both DNA repair pathway choice and transcription. Whereas ubiquitination is generally associated with protein degradation, the impact of sumoylation has been more mysterious. Sumoylation effects are largely mediated by the subnuclear localization of its targets, particularly in response to DNA damage. This is governed in part by the concentration of SUMO protease at nuclear pores (1,2). We review here the roles of sumoylation in determining subnuclear locus positioning relative to the nuclear envelope and the nuclear envelope to facilitate repair and to regulate transcription.

Keywords:  DNA-protein crosslink; STUbL; SUMOylation; damage relocation; double-strand break repair; proteolysis; replication fork collapse; telomeres; transcription

DOI:  https://doi.org/10.1002/1873-3468.14751
bioRxiv. 2023 Sep 27. pii: 2023.09.27.559793. [Epub ahead of print]

Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control.

Katharine Y Chen, Heungwon Park, Arvind Rasi Subramaniam.

Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae . In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level.

DOI: https://doi.org/10.1101/2023.09.27.559793
Nat Chem Biol. 2023 Oct 12.

Activation of human STING by a molecular glue-like compound.

Jie Li, Stephen M Canham, Hua Wu, Martin Henault, Lihao Chen, Guoxun Liu, Yu Chen, Gary Yu, Howard R Miller, Viktor Hornak, Scott M Brittain, Gregory A Michaud, Antonin Tutter, Wendy Broom, Mary Ellen Digan, Sarah M McWhirter, Kelsey E Sivick, Helen T Pham, Christine H Chen, George S Tria, Jeffery M McKenna, Markus Schirle, Xiaohong Mao, Thomas B Nicholson, Yuan Wang, Jeremy L Jenkins, Rishi K Jain, John A Tallarico, Sejal J Patel, Lianxing Zheng, Nathan T Ross, Charles Y Cho, Xuewu Zhang, Xiao-Chen Bai, Yan Feng.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

DOI: https://doi.org/10.1038/s41589-023-01434-y
iScience. 2023 Oct 20. 26(10): 107919

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint.

Kevin P Guay, Roberta Ibba, J L Kiappes, Snežana Vasiljević, Francesco Bonì, Maria De Benedictis, Ilaria Zeni, James D Le Cornu, Mario Hensen, Anu V Chandran, Anastassia L Kantsadi, Alessandro T Caputo, Juan I Blanco Capurro, Yusupha Bayo, Johan C Hill, Kieran Hudson, Andrea Lia, Juliane Brun, Stephen G Withers, Marcelo Martí, Emiliano Biasini, Angelo Santino, Matteo De Rosa, Mario Milani, Carlos P Modenutti, Daniel N Hebert, Nicole Zitzmann, Pietro Roversi.

  Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 "WY" conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 μM binding affinity for CtUGGTGT24in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 μM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.

Keywords:  Cell biology; Chemistry; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2023.107919
Nat Commun. 2023 Oct 12. 14(1): 6414

Proteogenetic drug response profiling elucidates targetable vulnerabilities of myelofibrosis.

Mattheus H E Wildschut, Julien Mena, Cyril Dördelmann, Marc van Oostrum, Benjamin D Hale, Jens Settelmeier, Yasmin Festl, Veronika Lysenko, Patrick M Schürch, Alexander Ring, Yannik Severin, Michael S Bader, Patrick G A Pedrioli, Sandra Goetze, Audrey van Drogen, Stefan Balabanov, Radek C Skoda, Massimo Lopes, Bernd Wollscheid, Alexandre P A Theocharides, Berend Snijder.

Myelofibrosis is a hematopoietic stem cell disorder belonging to the myeloproliferative neoplasms. Myelofibrosis patients frequently carry driver mutations in either JAK2 or Calreticulin (CALR) and have limited therapeutic options. Here, we integrate ex vivo drug response and proteotype analyses across myelofibrosis patient cohorts to discover targetable vulnerabilities and associated therapeutic strategies. Drug sensitivities of mutated and progenitor cells were measured in patient blood using high-content imaging and single-cell deep learning-based analyses. Integration with matched molecular profiling revealed three targetable vulnerabilities. First, CALR mutations drive BET and HDAC inhibitor sensitivity, particularly in the absence of high Ras pathway protein levels. Second, an MCM complex-high proliferative signature corresponds to advanced disease and sensitivity to drugs targeting pro-survival signaling and DNA replication. Third, homozygous CALR mutations result in high endoplasmic reticulum (ER) stress, responding to ER stressors and unfolded protein response inhibition. Overall, our integrated analyses provide a molecularly motivated roadmap for individualized myelofibrosis patient treatment.

DOI: https://doi.org/10.1038/s41467-023-42101-z
Mol Cell Proteomics. 2023 Oct 05. pii: S1535-9476(23)00168-8. [Epub ahead of print] 100657

Higher-order structural organization of the mitochondrial proteome charted by in situ cross-linking mass spectrometry.

Johannes F Hevler, Albert J R Heck.

  Mitochondria are densely packed with proteins, of which most are involved physically or more transiently in protein-protein interactions (PPIs). Mitochondria host among others all enzymes of the Krebs cycle and the oxidative phosphorylation (OXPHOS) pathway and are foremost associated with cellular bioenergetics (1, 2). However, mitochondria are also important contributors to apoptotic cell death (3) and contain their own genome (4) indicating that they play additionally an eminent role in processes beyond bioenergetics (5). Despite intense efforts in identifying and characterizing mitochondrial protein complexes by structural biology and proteomics techniques, many PPIs have remained elusive. Several of these (membrane embedded) PPIs are less stable in-vitro hampering their characterization by most contemporary methods in structural biology. Particularly in these cases, cross-linking mass spectrometry (XL-MS) has proven valuable for the in-depth characterization of mitochondrial protein complexes in situ. Here, we highlight experimental strategies for the analysis of proteome-wide protein-protein interactions in mitochondria using XL-MS. We showcase the ability of in situ XL-MS as a tool to map sub-organelle interactions and topologies, and aid in refining structural models of protein complexes. We describe some of the most recent technological advances in XL-MS that may benefit the in situ characterization of PPIs even further, especially when combined with electron microscopy and structural modelling.

Keywords:  In situ cross-linking mass spectrometry; cross-linking reagents; membrane complexes; mitochondria; protein localization; protein-protein interactions

DOI:  https://doi.org/10.1016/j.mcpro.2023.100657
Nature. 2023 Oct 11.

Apoptotic stress causes mtDNA release during senescence and drives the SASP.

Stella Victorelli, Hanna Salmonowicz, James Chapman, Helene Martini, Maria Grazia Vizioli, Joel S Riley, Catherine Cloix, Ella Hall-Younger, Jair Machado Espindola-Netto, Diana Jurk, Anthony B Lagnado, Lilian Sales Gomez, Joshua N Farr, Dominik Saul, Rebecca Reed, George Kelly, Madeline Eppard, Laura C Greaves, Zhixun Dou, Nicholas Pirius, Karolina Szczepanowska, Rebecca A Porritt, Huijie Huang, Timothy Y Huang, Derek A Mann, Claudio Akio Masuda, Sundeep Khosla, Haiming Dai, Scott H Kaufmann, Emmanouil Zacharioudakis, Evripidis Gavathiotis, Nathan K LeBrasseur, Xue Lei, Alva G Sainz, Viktor I Korolchuk, Peter D Adams, Gerald S Shadel, Stephen W G Tait, João F Passos.

Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP)1. Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated2. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die3. Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS-STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.

DOI: https://doi.org/10.1038/s41586-023-06621-4
Nat Commun. 2023 Oct 13. 14(1): 6429

Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility.

Georg Dorn, Christoph Gmeiner, Tebbe de Vries, Emil Dedic, Mihajlo Novakovic, Fred F Damberger, Christophe Maris, Esteban Finol, Chris P Sarnowski, Joachim Kohlbrecher, Timothy J Welsh, Sreenath Bolisetty, Raffaele Mezzenga, Ruedi Aebersold, Alexander Leitner, Maxim Yulikov, Gunnar Jeschke, Frédéric H-T Allain.

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

DOI: https://doi.org/10.1038/s41467-023-42012-z
iScience. 2023 Oct 20. 26(10): 107960

Lipid droplets modulate proteostasis, SQST-1/SQSTM1 dynamics, and lifespan in C. elegans.

Anita V Kumar, Joslyn Mills, Wesley M Parker, Joshua A Leitão, Diego I Rodriguez, Sandrine E Daigle, Celeste Ng, Rishi Patel, Joseph L Aguilera, Joseph R Johnson, Shi Quan Wong, Louis R Lapierre.

  In several long-lived Caenorhabditis elegans strains, such as insulin/IGF-1 receptor daf-2 mutants, enhanced proteostatic mechanisms are accompanied by elevated intestinal lipid stores, but their role in longevity is unclear. Here, while determining the regulatory network of the selective autophagy receptor SQST-1/SQSTM1, we uncovered an important role for lipid droplets in proteostasis and longevity. Using genome-wide RNAi screening, we identified several SQST-1 modulators, including lipid droplets-associated and aggregation-prone proteins. Expansion of intestinal lipid droplets by silencing the conserved cytosolic triacylglycerol lipase gene atgl-1/ATGL enhanced autophagy, and extended lifespan. Notably, a substantial amount of ubiquitinated proteins were found on lipid droplets. Reducing lipid droplet levels exacerbated the proteostatic collapse when autophagy or proteasome function was compromised, and significantly reduced the lifespan of long-lived daf-2 animals. Altogether, our study uncovered a key role for lipid droplets in C. elegans as a proteostatic mediator that modulates ubiquitinated protein accumulation, facilitates autophagy, and promotes longevity.

Keywords:  Biochemistry; Biological sciences

DOI:  https://doi.org/10.1016/j.isci.2023.107960