bims-proteo 2022-12-18 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022–12–18
33 papers selected by
Eric Chevet, INSERM

In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay.
CHIP: A Co-chaperone for Degradation by the Proteasome and Lysosome.
Co-chaperones of the Human Endoplasmic Reticulum: An Update.
Protein biosynthesis at the ER: finding the right accessories.
Impaired ribosome-associated quality control of C9orf72 arginine-rich dipeptide-repeat proteins.
CRL2ZER1/ZYG11B recognizes small N-terminal residues for degradation.
Specification of Hsp70 Function by Hsp40 Co-chaperones.
Mutant MESD links cellular stress to type I collagen aggregation in osteogenesis imperfecta type XX.
Endoplasmic reticulum protein 5 attenuates platelet endoplasmic reticulum stress and secretion in a mouse model.
NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy.
Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress.
An ERAD-related E2-E3 enzyme pair controls grain size and weight through the brassinosteroid signaling pathway in rice.
Membrane curvature sensing and stabilization by the autophagic LC3 lipidation machinery.
GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum.
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response.
Endosomal lipid signaling reshapes the endoplasmic reticulum to control mitochondrial function.
The pleiotropic roles of eIF5A in cellular life and its therapeutic potential in cancer.
Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.
The Alarmone Diadenosine Tetraphosphate as a Cosubstrate for Protein AMPylation.
The hepatic integrated stress response suppresses the somatotroph axis to control liver damage in nonalcoholic fatty liver disease.
The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response.
A male germ-cell-specific ribosome controls male fertility.
Emerging Proteolysis Targeting Antibodies (PROTABs) for Application in Cancer Therapy.
ATG9A prevents TNF cytotoxicity by an unconventional lysosomal targeting pathway.
Two isoleucyl tRNAs that decode synonymous codons divergently regulate breast cancer metastatic growth by controlling translation of proliferation-regulating genes.
Real-time single-molecule observation of chaperone-assisted protein folding.
Hepatic neddylation deficiency triggers fatal liver injury via inducing NF-κB-inducing kinase in mice.
Structure-Based Discovery of Selective Histone Deacetylase 8 Degraders with Potent Anticancer Activity.
Inositol-requiring enzyme 1α/X-box protein 1 pathway expression is impaired in pediatric cholestatic liver disease explants.
Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER.
FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast.
Small-molecule PROTAC mediates targeted protein degradation to treat STAT3-dependent epithelial cancer.
Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis.

Bio Protoc. 2022 Nov 05. pii: e4547. [Epub ahead of print]12(21):

In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay.

Rebecca J Burge, Katie H Jameson, Anthony J Wilkinson, Jeremy C Mottram.

  Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana , specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues.

Keywords:   Chain formation ; E2 conjugating ; E3 ligase ; Ubiquitin ; Ubiquitination

DOI:  https://doi.org/10.21769/BioProtoc.4547
Subcell Biochem. 2023 ;101 351-387

CHIP: A Co-chaperone for Degradation by the Proteasome and Lysosome.

Abantika Chakraborty, Adrienne L Edkins.

  Protein homeostasis relies on a balance between protein folding and protein degradation. Molecular chaperones like Hsp70 and Hsp90 fulfill well-defined roles in protein folding and conformational stability via ATP-dependent reaction cycles. These folding cycles are controlled by associations with a cohort of non-client protein co-chaperones, such as Hop, p23, and Aha1. Pro-folding co-chaperones facilitate the transit of the client protein through the chaperone-mediated folding process. However, chaperones are also involved in proteasomal and lysosomal degradation of client proteins. Like folding complexes, the ability of chaperones to mediate protein degradation is regulated by co-chaperones, such as the C-terminal Hsp70-binding protein (CHIP/STUB1). CHIP binds to Hsp70 and Hsp90 chaperones through its tetratricopeptide repeat (TPR) domain and functions as an E3 ubiquitin ligase using a modified RING finger domain (U-box). This unique combination of domains effectively allows CHIP to network chaperone complexes to the ubiquitin-proteasome and autophagosome-lysosome systems. This chapter reviews the current understanding of CHIP as a co-chaperone that switches Hsp70/Hsp90 chaperone complexes from protein folding to protein degradation.

Keywords:  Autophagy; CHIP; Co-chaperones; Lysosome; Proteasome; STUB1; Ubiquitin

DOI:  https://doi.org/10.1007/978-3-031-14740-1_12
Subcell Biochem. 2023 ;101 247-291

Co-chaperones of the Human Endoplasmic Reticulum: An Update.

Armin Melnyk, Sven Lang, Mark Sicking, Richard Zimmermann, Martin Jung.

  In mammalian cells, the rough endoplasmic reticulum (ER) plays central roles in the biogenesis of extracellular plus organellar proteins and in various signal transduction pathways. For these reasons, the ER comprises molecular chaperones, which are involved in import, folding, assembly, export, plus degradation of polypeptides, and signal transduction components, such as calcium channels, calcium pumps, and UPR transducers plus adenine nucleotide carriers/exchangers in the ER membrane. The calcium- and ATP-dependent ER lumenal Hsp70, termed immunoglobulin heavy-chain-binding protein or BiP, is the central player in all these activities and involves up to nine different Hsp40-type co-chaperones, i.e., ER membrane integrated as well as ER lumenal J-domain proteins, termed ERj or ERdj proteins, two nucleotide exchange factors or NEFs (Grp170 and Sil1), and NEF-antagonists, such as MANF. Here we summarize the current knowledge on the ER-resident BiP/ERj chaperone network and focus on the interaction of BiP with the polypeptide-conducting and calcium-permeable Sec61 channel of the ER membrane as an example for BiP action and how its functional cycle is linked to ER protein import and various calcium-dependent signal transduction pathways.

Keywords:  ATP/ADP exchange; Calcium-dependent signal transduction; Cellular calcium homeostasis; ER calcium leakage; ER energy homeostasis; ER protein import; ER-associated protein degradation; Human endoplasmic reticulum; J-domain proteins; Protein folding

DOI:  https://doi.org/10.1007/978-3-031-14740-1_9
Mol Biol Cell. 2023 Jan 01. 34(1): pe1

Protein biosynthesis at the ER: finding the right accessories.

Sichen Shao.

More than 30% of eukaryotic proteins contain domains that must translocate across or integrate into the endoplasmic reticulum (ER) membrane. With few exceptions, protein translocation and transmembrane domain integration at the ER require the conserved Sec61 translocon. Decades of studies have established a clear mechanistic model for how the Sec61 translocon functions. The biosynthesis of distinct subsets of proteins at the ER also involves accessory factors that interact with the Sec61 translocon and translocating nascent proteins. However, assigning specific functions to many translocon accessory factors has been a persistent challenge in the field. This Perspective discusses recent insights into mechanisms that promote protein biosynthesis at the ER through accessory factors that directly regulate the Sec61 translocon or chaperone nascent proteins within the ER membrane. These translocon accessory factor functions, and more still to be discovered, are essential for producing a diverse and high-fidelity proteome at the ER.

DOI: https://doi.org/10.1091/mbc.E21-09-0451
Brain. 2022 Dec 14. pii: awac479. [Epub ahead of print]

Impaired ribosome-associated quality control of C9orf72 arginine-rich dipeptide-repeat proteins.

Ashley P Viera Ortiz, Gregory Cajka, Olamide A Olatunji, Bailey Mikytuck, Ophir Shalem, Edward B Lee.

  Protein quality control pathways have evolved to ensure the fidelity of protein synthesis and efficiently clear potentially toxic protein species. Defects in ribosome-associated quality control and its associated factors have been implicated in the accumulation of aberrant proteins and neurodegeneration. C9orf72 repeat-associated non-AUG translation has been suggested to involve inefficient translation elongation, lead to ribosomal pausing and activation of ribosome-associated quality control pathways. However, the role of the ribosome-associated quality control complex in the processing of proteins generated through this non-canonical translation is not well understood. Here we utilize reporter constructs containing the C9orf72-associated hexanucleotide repeat, ribosome-associated quality control complex deficient cell models and stain for ribosome-associated quality control markers in C9orf72-expansion carrier human tissue to understand its role in dipeptide repeat protein pathology. Our studies show that canonical ribosome-associated quality control substrates products are efficiently cleared by the ribosome-associated quality control complex in mammalian cells. Furthermore, using stalling reporter constructs, we show that repeats associated with the C9orf72-expansion induce ribosomal stalling when arginine (R)-rich dipeptide-repeat proteins are synthesized in a length-dependent manner. However, despite triggering this pathway, these arginine-rich dipeptide-repeat proteins are not efficiently processed by the core components of the ribosome-associated quality control complex (listerin, nuclear export mediator factor (NEMF) and valosin containing protein (VCP)) partly due to lack of lysine residues which precludes ubiquitination. Deficient processing by this complex may be implicated in C9orf72-expansion associated disease as dipeptide-repeat protein inclusions were observed to be predominantly devoid of ubiquitin and co-localize with NEMF in mutation carriers' frontal cortex and cerebellum tissue. These findings suggest that impaired processing of these arginine-rich dipeptide-repeat proteins derived from repeat-associated non-AUG translation by the ribosome-associated quality control complex may contribute to protein homeostasis dysregulation observed in C9orf72-expansion amyotrophic lateral sclerosis and frontotemporal degeneration neuropathogenesis.

Keywords:   C9orf72 ; ALS/FTD; NEMF; RAN translation; ribosome-associated quality control

DOI:  https://doi.org/10.1093/brain/awac479
Nat Commun. 2022 Dec 10. 13(1): 7636

CRL2ZER1/ZYG11B recognizes small N-terminal residues for degradation.

Yao Li, Yueling Zhao, Xiaojie Yan, Chen Ye, Sara Weirich, Bing Zhang, Xiaolu Wang, Lili Song, Chenhao Jiang, Albert Jeltsch, Cheng Dong, Wenyi Mi.

N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 binds better to Nt-Ser, -Ala, -Thr and -Cys than to -Gly, while ZYG11B prefers Nt-Gly but also has the capacity to recognize Nt-Ser, -Ala and -Cys in vitro. We found that Nt-Ser, -Ala and -Cys undergo Nt-acetylation catalyzed by Nt-acetyltransferase (NAT), thereby shielding them from recognition by ZER1/ZYG11B in cells. Instead, ZER1/ZYG11B readily targets a selection of small Nt-residues lacking Nt-acetylation for degradation in NAT-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.

DOI: https://doi.org/10.1038/s41467-022-35169-6
Subcell Biochem. 2023 ;101 127-139

Specification of Hsp70 Function by Hsp40 Co-chaperones.

Douglas M Cyr, Carlos H Ramos.

  Cellular homeostasis and stress survival requires maintenance of the proteome and suppression of proteotoxicity. Molecular chaperones promote cell survival through repair of misfolded proteins and cooperation with protein degradation machines to discard terminally damaged proteins. Hsp70 family members play an essential role in cellular protein metabolism by binding and releasing non-native proteins to facilitate protein folding, refolding, and degradation. Hsp40 (DnaJ-like proteins) family members are Hsp70 co-chaperones that determine the fate of Hsp70 clients by facilitating protein folding, assembly, and degradation. Hsp40s select substrates for Hsp70 via use of an intrinsic chaperone activity to bind non-native regions of proteins. During delivery of bound cargo Hsp40s employ a conserved J-domain to stimulate Hsp70 ATPase activity and thereby stabilize complexes between Hsp70 and non-native proteins. This review describes the mechanisms by which different Hsp40s use specialized sub-domains to direct clients of Hsp70 for triage between folding versus degradation.

Keywords:  Hsp40; Hsp70; Molecular chaperone; Protein folding; Protein triage

DOI:  https://doi.org/10.1007/978-3-031-14740-1_4
Matrix Biol. 2022 Dec 13. pii: S0945-053X(22)00148-2. [Epub ahead of print]

Mutant MESD links cellular stress to type I collagen aggregation in osteogenesis imperfecta type XX.

Debasish Kumar Ghosh, Prajna Udupa, Akshaykumar Nanaji Shrikondawar, Gandham SriLakshmi Bhavani, Hitesh Shah, Akash Ranjan, Katta M Girisha.

  Aberrant forms of endoplasmic reticulum (ER)-resident chaperones are implicated in loss of protein quality control in rare diseases. Here we report a novel mutation (p.Asp233Asn) in the ER retention signal of MESD by whole exome sequencing of an individual diagnosed with osteogenesis imperfecta (OI) type XX. While MESDD233N has similar stability and chaperone activity as wild-type MESD, its mislocalization to cytoplasm leads to imbalance of ER proteostasis, resulting in improper folding and aggregation of proteins, including LRP5 and type I collagen. Aggregated LRP5 loses its plasma membrane localization to disrupt the expression of WNT-responsive genes, such as BMP2, BMP4, in proband fibroblasts. We show that MESD is a direct chaperone of pro-α1(I) [COL1A1], and absence of MESDD233N in ER results in cytosolic type I collagen aggregates that remain mostly not secreted. While cytosolic type I collagen aggregates block the intercellular nanotubes, decreased extracellular type I collagen also results in loss of interaction of ITGB1 with type I collagen and weaker attachment of fibroblasts to matrix. Although proband fibroblasts show increased autophagy to degrade the aggregated type I collagen, an overall cellular stress overwhelms the proband fibroblasts. In summary, we present an essential chaperone function of MESD for LRP5 and type I collagen and demonstrating how the D233N mutation in MESD correlates with impaired WNT signaling and proteostasis in OI.

Keywords:  Autophagy; MESD; Osteogenesis imperfecta; Protein aggregation; Proteostasis imbalance; Type I collagen

DOI:  https://doi.org/10.1016/j.matbio.2022.12.001
Blood Adv. 2022 Dec 12. pii: bloodadvances.2022008457. [Epub ahead of print]

Endoplasmic reticulum protein 5 attenuates platelet endoplasmic reticulum stress and secretion in a mouse model.

Angelina J Lay, Alexander Dupuy, Lejla Hagimola, Jessica Tieng, Mark Larance, Yunwei Zhang, Jean Yang, Yvonne Xiangyue Kong, Joyce Chiu, Emilia Charlotte Gray, Zihao Qin, Diana Schmidt, Jessica A A Maclean, Benjamin Rink Hofma, Marc L Ellis, Maggie L Kalev-Zylinska, Yair Argon, Shaun P Jackson, Philip Hogg, Freda H Passam.

Extracellular protein disulfide isomerases (PDIs), including PDI, endoplasmic reticulum protein 57 (ERp57), ERp72, ERp46 and ERp5, are required for in vivo thrombus formation in mice. Platelets secrete PDIs upon activation, which regulate platelet aggregation. However, platelets secrete only ~10% of their PDI content extracellularly. The intracellular role of PDIs in platelet function is unknown. In the current study, we aimed to characterize the role of ERp5 (gene Pdia6) using platelet conditional knockout mice, platelet factor 4 (Pf4) Cre+/ERp5fl/fl. Pf4Cre+/ERp5fl/fl mice developed mild macrothrombocytopenia. Platelets deficient in ERp5 showed marked dysregulation of their ER, indicated by a 2-fold upregulation of ER proteins, including PDI, ERp57, ERp72, ERp46, 78 kDa glucose-regulated protein (GRP78) and calreticulin. ERp5 deficient platelets showed an enhanced ER stress response to ex vivo and in vivo ER stress inducers, with enhanced phosphorylation of eukaryotic translation initiation factor 2A (eIF2a) and inositol-requiring enzyme 1 (IRE1). ERp5 deficiency was associated with increased secretion of PDIs, an enhanced response to thromboxane A2 (TXA2) receptor activation, and increased thrombus formation in vivo. Our results support that ERp5 acts as negative regulator of ER stress responses in platelets, and highlights the importance of a disulfide isomerase in platelet ER homeostasis. The results also indicate a previously unanticipated role of platelet ER stress in platelet secretion and thrombosis. This may have important implications for therapeutic applications of ER stress inhibitors in thrombosis.

DOI: https://doi.org/10.1182/bloodadvances.2022008457
EMBO J. 2022 Dec 14. e111372

NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy.

Tetsushi Kataura, Elsje G Otten, Yoana Rabanal-Ruiz, Elias Adriaenssens, Francesca Urselli, Filippo Scialo, Lanyu Fan, Graham R Smith, William M Dawson, Xingxiang Chen, Wyatt W Yue, Agnieszka K Bronowska, Bernadette Carroll, Sascha Martens, Michael Lazarou, Viktor I Korolchuk.

  Mitophagy, the elimination of mitochondria via the autophagy-lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin-dependent mitophagy. We identified redox-sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.

Keywords:  NDP52; autophagy; mitophagy; p62; redox

DOI:  https://doi.org/10.15252/embj.2022111372
Proc Natl Acad Sci U S A. 2022 Dec 20. 119(51): e2214957119

Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress.

Hui-Hsing Hung, Yasuko Nagatsuka, Tatiana Soldà, Vamsi K Kodali, Kazuhisa Iwabuchi, Hiroyuki Kamiguchi, Koki Kano, Ichiro Matsuo, Kazutaka Ikeda, Randal J Kaufman, Maurizio Molinari, Peter Greimel, Yoshio Hirabayashi.

  Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

Keywords:  CHOP; UGGT; hypoxia; phosphatidylglucoside; saturated lipid

DOI:  https://doi.org/10.1073/pnas.2214957119
Plant Cell. 2022 Dec 15. pii: koac364. [Epub ahead of print]

An ERAD-related E2-E3 enzyme pair controls grain size and weight through the brassinosteroid signaling pathway in rice.

Jing Li, Baolan Zhang, Penggen Duan, Li Yan, Haiyue Yu, Limin Zhang, Na Li, Leiying Zheng, Tuanyao Chai, Ran Xu, Yunhai Li.

  Grain size is an important agronomic trait, but our knowledge about grain size determination in crops is still limited. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a special ubiquitin proteasome system that is involved in degrading misfolded or incompletely folded proteins in the ER. Here, we report that SMALL GRAIN 3 (SMG3) and DECREASED GRAIN SIZE 1 (DGS1), an ERAD-related E2-E3 enzyme pair, regulate grain size and weight through the brassinosteroid (BR) signaling pathway in rice (Oryza sativa). SMG3 encodes a homolog of Arabidopsis (Arabidopsis thaliana) UBIQUITIN CONJUGATING ENZYME 32 (UBC32), which is a conserved ERAD-associated E2 ubiquitin conjugating enzyme. SMG3 interacts with another grain size regulator, DGS1. Loss of function of SMG3 or DGS1 results in small grains, while overexpression of SMG3 or DGS1 leads to long grains. Further analyses showed that DGS1 is an active E3 ubiquitin ligase and co-locates with SMG3 in the ER. SMG3 and DGS1 are involved in BR signaling. DGS1 ubiquitinates the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and affects its accumulation. Genetic analysis suggests that SMG3, DGS1 and BRI1 act together to regulate grain size and weight. In summary, our findings identify an ERAD-related E2-E3 pair that regulates grain size and weight, which gives insight into the function of ERAD in grain size control and BR signaling.

Keywords:  BR signaling; DGS1; ERAD; SMG3/UBC32; grain size

DOI:  https://doi.org/10.1093/plcell/koac364
Sci Adv. 2022 Dec 14. 8(50): eadd1436

Membrane curvature sensing and stabilization by the autophagic LC3 lipidation machinery.

Liv E Jensen, Shanlin Rao, Martina Schuschnig, A King Cada, Sascha Martens, Gerhard Hummer, James H Hurley.

How the highly curved phagophore membrane is stabilized during autophagy initiation is a major open question in autophagosome biogenesis. Here, we use in vitro reconstitution on membrane nanotubes and molecular dynamics simulations to investigate how core autophagy proteins in the LC3 (Microtubule-associated proteins 1A/1B light chain 3) lipidation cascade interact with curved membranes, providing insight into their possible roles in regulating membrane shape during autophagosome biogenesis. ATG12(Autophagy-related 12)-ATG5-ATG16L1 was up to 100-fold enriched on highly curved nanotubes relative to flat membranes. At high surface density, ATG12-ATG5-ATG16L1 binding increased the curvature of the nanotubes. While WIPI2 (WD repeat domain phosphoinositide-interacting protein 2) binding directs membrane recruitment, the amphipathic helix α2 of ATG16L1 is responsible for curvature sensitivity. Molecular dynamics simulations revealed that helix α2 of ATG16L1 inserts shallowly into the membrane, explaining its curvature-sensitive binding to the membrane. These observations show how the binding of the ATG12-ATG5-ATG16L1 complex to the early phagophore rim could stabilize membrane curvature and facilitate autophagosome growth.

DOI: https://doi.org/10.1126/sciadv.add1436
EMBO J. 2022 Dec 16. e111513

GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum.

Zhi-Ping Qiu, Zi-Cun Lin, Ao Hu, Yuan-Bin Liu, Wan-Er Zeng, Xiaolu Zhao, Xiong-Jie Shi, Jie Luo, Bao-Liang Song.

  Hedgehog (Hh) signaling pathway plays a pivotal role in embryonic development. Hh binding to Patched1 (PTCH1) derepresses Smoothened (SMO), thereby activating the downstream signal transduction. Covalent SMO modification by cholesterol in its cysteine-rich domain (CRD) is essential for SMO function. SMO cholesterylation is a calcium-accelerated autoprocessing reaction, and STIM1-ORAI1-mediated store-operated calcium entry promotes cholesterylation and activation of endosome-localized SMO. However, it is unknown whether the Hh-PTCH1 interplay regulates the activity of the endoplasmic reticulum (ER)-localized SMO. Here, we found that PTCH1 inhibited the COPII-dependent export of SMO from the ER, whereas Hh promoted this process. The RRxWxR amino acid motif in the cytosolic tail of SMO was essential for COPII recognition, ciliary localization, and signal transduction activity. Hh and PTCH1 regulated cholesterol modification of the ER-localized SMO, and SMO cholesterylation accelerated its exit from ER. The GRAMD1/ASTER sterol transport proteins facilitated cholesterol transfer to ER from PM, resulting in increased SMO cholesterylation and enhanced Hh signaling. Collectively, we reveal a regulatory role of GRAMD-mediated cholesterol transport in ER-resident SMO maturation and Hh signaling.

Keywords:  COPII; GRAMD1s; SMO; cholesterylation; glycosylation

DOI:  https://doi.org/10.15252/embj.2022111513
EMBO Rep. 2022 Dec 14. e54844

K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response.

Takahisa Kouwaki, Tasuku Nishimura, Guanming Wang, Reiko Nakagawa, Hiroyuki Oshiumi.

  Type I interferons (IFNs) exhibit strong antiviral activity and induce the expression of antiviral proteins. Since excessive expression of type I IFNs is harmful to the host, their expression should be turned off at the appropriate time. In this study, we find that post-translational modification of LGP2, a member of the RIG-I-like receptor family, modulates antiviral innate immune responses. The LGP2 protein undergoes K63-linked polyubiquitination in response to cytoplasmic double-stranded RNAs or viral infection. Our mass spectrometry analysis reveals the K residues ubiquitinated by the Riplet ubiquitin ligase. LGP2 ubiquitination occurs with a delay compared to RIG-I ubiquitination. Interestingly, ubiquitination-defective LGP2 mutations increase the expression of type I IFN at a late phase, whereas the mutant proteins attenuate other antiviral proteins, such as SP100, PML, and ANKRD1. Our data indicate that delayed polyubiquitination of LGP2 fine-tunes RIG-I-dependent antiviral innate immune responses at a late phase of viral infection.

Keywords:  LGP2; innate immunity; ubiquitination; virus

DOI:  https://doi.org/10.15252/embr.202254844
Science. 2022 Dec 16. 378(6625): eabq5209

Endosomal lipid signaling reshapes the endoplasmic reticulum to control mitochondrial function.

Wonyul Jang, Dmytro Puchkov, Paula Samsó, YongTian Liang, Michal Nadler-Holly, Stephan J Sigrist, Ulrich Kintscher, Fan Liu, Kamel Mamchaoui, Vincent Mouly, Volker Haucke.

Cells respond to fluctuating nutrient supply by adaptive changes in organelle dynamics and in metabolism. How such changes are orchestrated on a cell-wide scale is unknown. We show that endosomal signaling lipid turnover by MTM1, a phosphatidylinositol 3-phosphate [PI(3)P] 3-phosphatase mutated in X-linked centronuclear myopathy in humans, controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes, resulting in the conversion of ER tubules into sheets, the inhibition of mitochondrial fission, and sustained oxidative metabolism. Our results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to fluctuating nutrient environments.

DOI: https://doi.org/10.1126/science.abq5209
Biochem Soc Trans. 2022 Dec 16. 50(6): 1885-1895

The pleiotropic roles of eIF5A in cellular life and its therapeutic potential in cancer.

Aristeidis Panagiotis Sfakianos, Rebecca Mallory Raven, Anne Elizabeth Willis.

  Protein synthesis is dysregulated in the majority of cancers and this process therefore provides a good therapeutic target. Many novel anti-cancer agents are directed to target the initiation stage of translation, however, translation elongation also holds great potential as a therapeutic target. The elongation factor eIF5A that assists the formation of peptidyl bonds during the elongation process is of considerable interest in this regard. Overexpression of eIF5A has been linked with the development of a variety of cancers and inhibitors of the molecule have been proposed for anti-cancer clinical applications. eIF5A is the only protein in the cell that contains the post-translational modification hypusine. Hypusination is a two-step enzymatic process catalysed by the Deoxyhypusine Synthase (DHPS) and Deoxyhypusine Hydroxylase (DOHH). In addition, eIF5A can be acetylated by p300/CBP-associated factor (PCAF) which leads to translocation of the protein to the nucleus and its deactivation. In addition to the nucleus, eIF5A has been found in the mitochondria and the endoplasmic reticulum (ER) with eIF5A localisation related to function from regulation of mitochondrial activity and apoptosis to maintenance of ER integrity and control of the unfolded protein response (UPR). Given the pleiotropic functions of eIF5A and by extension the hypusination enzymes, this system is being considered as a target for a range of cancers including multiple myeloma, B-Cell lymphoma, and neuroblastoma. In this review, we explore the role of eIF5A and discuss the therapeutic strategies that are currently developing both in the pre- and the clinical stage.

Keywords:  DHPS; DOHH; cancer; eIF5A; hypusination; translation

DOI:  https://doi.org/10.1042/BST20221035
Sci Adv. 2022 Dec 14. 8(50): eabq2216

Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

Thang Cong Do, Jun Wei Lau, Caixia Sun, Songhan Liu, Khoa Tuan Kha, Seok Ting Lim, Yu Yang Oon, Yuet Ping Kwan, Jia Jia Ma, Yuguang Mu, Xiaogang Liu, Thomas James Carney, Xiaomeng Wang, Bengang Xing.

Epigenetic mediation through bromodomain and extraterminal (BET) proteins have progressively translated protein imbalance into effective cancer treatment. Perturbation of druggable BET proteins through proteolysis-targeting chimeras (PROTACs) has recently contributed to the discovery of effective therapeutics. Unfortunately, precise and microenvironment-activatable BET protein degradation content with promising tumor selectivity and pharmacological suitability remains elusive. Here, we present an enzyme-derived clicking PROTACs (ENCTACs) capable of orthogonally cross-linking two disparate small-molecule warhead ligands that recognize BET bromodomain-containing protein 4 (BRD4) protein and E3 ligase within tumors only upon hypoxia-induced activation of nitroreductase enzyme. This localized formation of heterobifunctional degraders promotes specific down-regulation of BRD4, which subsequently alters expression of epigenetic targets and, therefore, allows precise modulation of hypoxic signaling in live cells, zebrafish, and living mice with solid tumors. Our activation-feedback system demonstrates compelling superiorities and may enable the PROTAC technology with more flexible practicality and druggable potency for precision medicine in the near future.

DOI: https://doi.org/10.1126/sciadv.abq2216
Angew Chem Int Ed Engl. 2022 Dec 16.

The Alarmone Diadenosine Tetraphosphate as a Cosubstrate for Protein AMPylation.

Matthias Frese, Philip Saumer, Yizhi Yuan, Doreen Herzog, Dorothea Höpfner, Aymelt Itzen, Andreas Marx.

  Diadenosine polyphosphates (ApnAs) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed ApnAs for their usage as cosubstrate for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as cosubstrate is a response to ER stress. Here, we demonstrate that Ap4A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap4A levels during cellular stress differs from when Ap4A is present at low concentrations allowing response to extracellular cues.

Keywords:  AMPylation; Activity-based protein profiling; Post-translational modification; chemical proteomics; nucleotides

DOI:  https://doi.org/10.1002/anie.202213279
Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01691-6. [Epub ahead of print]41(11): 111803

The hepatic integrated stress response suppresses the somatotroph axis to control liver damage in nonalcoholic fatty liver disease.

Rika Ohkubo, Wei-Chieh Mu, Chih-Ling Wang, Zehan Song, Marine Barthez, Yifei Wang, Nathaniel Mitchener, Rasul Abdullayev, Yeong Rim Lee, Yuze Ma, Megan Curtin, Suraj Srinivasan, Xingjia Zhang, Fanghan Yang, Peter H Sudmant, Angela Oliveira Pisco, Norma Neff, Cole M Haynes, Danica Chen.

  Nonalcoholic fatty liver disease (NAFLD) can be ameliorated by calorie restriction, which leads to the suppressed somatotroph axis. Paradoxically, the suppressed somatotroph axis is associated with patients with NAFLD and is correlated with the severity of fibrosis. How the somatotroph axis becomes dysregulated and whether the repressed somatotroph axis impacts liver damage during the progression of NAFLD are unclear. Here, we identify a regulatory branch of the hepatic integrated stress response (ISR), which represses the somatotroph axis in hepatocytes through ATF3, resulting in enhanced cell survival and reduced cell proliferation. In mouse models of NAFLD, the ISR represses the somatotroph axis, leading to reduced apoptosis and inflammation but decreased hepatocyte proliferation and exacerbated fibrosis in the liver. NAD+ repletion reduces the ISR, rescues the dysregulated somatotroph axis, and alleviates NAFLD. These results establish that the hepatic ISR suppresses the somatotroph axis to control cell fate decisions and liver damage in NAFLD.

Keywords:  ATF3; CP: Metabolism; ER stress; IGF-1; SIRT7; aging; integrated stress response; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis; sirtuin; somatotroph axis

DOI:  https://doi.org/10.1016/j.celrep.2022.111803
Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01677-1. [Epub ahead of print]41(11): 111789

The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response.

Douja Chamseddine, Siraje A Mahmud, Aundrea K Westfall, Todd A Castoe, Rance E Berg, Mark W Pellegrino.

  Organisms use several strategies to mitigate mitochondrial stress, including the activation of the mitochondrial unfolded protein response (UPRmt). The UPRmt in Caenorhabditis elegans, regulated by the transcription factor ATFS-1, expands on this recovery program by inducing an antimicrobial response against pathogens that target mitochondrial function. Here, we show that the mammalian ortholog of ATFS-1, ATF5, protects the host during infection with enteric pathogens but, unexpectedly, by maintaining the integrity of the intestinal barrier. Intriguingly, ATF5 supports intestinal barrier function by promoting a satiety response that prevents obesity and associated hyperglycemia. This consequently averts dysregulated glucose metabolism that is detrimental to barrier function. Mechanistically, we show that intestinal ATF5 stimulates the satiety response by transcriptionally regulating the gastrointestinal peptide hormone cholecystokinin, which promotes the secretion of the hormone leptin. We propose that ATF5 protects the host from enteric pathogens by promoting intestinal barrier function through a satiety-response-mediated metabolic control mechanism.

Keywords:  ATF5; CP: Metabolism; CP: Molecular biology; UPR(mt); cholecystokinin; colitis; epithelial barrier; host-pathogen interaction; hyperglycemia; leptin; mitochondria; satiety

DOI:  https://doi.org/10.1016/j.celrep.2022.111789
Nature. 2022 Dec 14.

A male germ-cell-specific ribosome controls male fertility.

Huiling Li, Yangao Huo, Xi He, Liping Yao, Hao Zhang, Yiqiang Cui, Huijuan Xiao, Wenxiu Xie, Dejiu Zhang, Yue Wang, Shu Zhang, Haixia Tu, Yiwei Cheng, Yueshuai Guo, Xintao Cao, Yunfei Zhu, Tao Jiang, Xuejiang Guo, Yan Qin, Jiahao Sha.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

DOI: https://doi.org/10.1038/s41586-022-05508-0
ACS Med Chem Lett. 2022 Dec 08. 13(12): 1833-1834

Emerging Proteolysis Targeting Antibodies (PROTABs) for Application in Cancer Therapy.

Robert B Kargbo.

The emerging proteolysis targeting antibodies (PROTABs) offer an attractive technology that circumvents some of the challenges of small-molecule intracellular degraders with limited bioavailability and cell permeability. PROTABs present an improved approach to target degradation of membrane-bound and cell surface proteins and use multispecific binding proteins such as multispecific antibodies that bind to at least one transmembrane E3 ubiquitin ligase as well as cell surface proteins intended for degradation.

DOI: https://doi.org/10.1021/acsmedchemlett.2c00458
Science. 2022 Dec 16. 378(6625): 1201-1207

ATG9A prevents TNF cytotoxicity by an unconventional lysosomal targeting pathway.

Jon Huyghe, Dario Priem, Lisette Van Hove, Barbara Gilbert, Jürgen Fritsch, Yasuo Uchiyama, Esther Hoste, Geert van Loo, Mathieu J M Bertrand.

Cell death induced by tumor necrosis factor (TNF) can be beneficial during infection by helping to mount proper immune responses. However, TNF-induced death can also drive a variety of inflammatory pathologies. Protectives brakes, or cell-death checkpoints, normally repress TNF cytotoxicity to protect the organism from its potential detrimental consequences. Thus, although TNF can kill, this only occurs when one of the checkpoints is inactivated. Here, we describe a checkpoint that prevents apoptosis through the detoxification of the cytotoxic complex IIa that forms upon TNF sensing. We found that autophagy-related 9A (ATG9A) and 200kD FAK family kinase-interacting protein (FIP200) promote the degradation of this complex through a light chain 3 (LC3)-independent lysosomal targeting pathway. This detoxification mechanism was found to counteract TNF receptor 1 (TNFR1)-mediated embryonic lethality and inflammatory skin disease in mouse models.

DOI: https://doi.org/10.1126/science.add6967
Nat Cancer. 2022 Dec 12.

Two isoleucyl tRNAs that decode synonymous codons divergently regulate breast cancer metastatic growth by controlling translation of proliferation-regulating genes.

Lisa B Earnest-Noble, Dennis Hsu, Siyu Chen, Hosseinali Asgharian, Mandayam Nandan, Maria C Passarelli, Hani Goodarzi, Sohail F Tavazoie.

The human genome contains 61 codons encoding 20 amino acids. Synonymous codons representing a given amino acid are decoded by a set of transfer RNAs (tRNAs) called isoacceptors. We report the surprising observation that two isoacceptor tRNAs that decode synonymous codons become modulated in opposing directions during breast cancer progression. Specifically, tRNAIleUAU became upregulated, whereas tRNAIleGAU became repressed as breast cancer cells attained enhanced metastatic capacity. Functionally, tRNAIleUAU promoted and tRNAIleGAU suppressed metastatic colonization in mouse xenograft models. These tRNAs mediated opposing effects on codon-dependent translation of growth-promoting genes, consistent with genomic enrichment or depletion of their cognate codons in mitotic genes. Our findings uncover a specific isoacceptor tRNA pair that act in opposition, divergently impacting growth-regulating genes and a disease phenotype. Degeneracy of the genetic code can thus be biologically exploited by human cancer cells via tRNA isoacceptor shifts that causally facilitate the transition toward a growth-promoting state.

DOI: https://doi.org/10.1038/s43018-022-00469-9
Sci Adv. 2022 Dec 14. 8(50): eadd0922

Real-time single-molecule observation of chaperone-assisted protein folding.

Nicholas R Marzano, Bishnu P Paudel, Antoine M van Oijen, Heath Ecroyd.

The ability of heat shock protein 70 (Hsp70) molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes toward efficient protein folding. Here, we used total internal reflection fluorescence (TIRF) microscopy and single-molecule fluorescence resonance energy transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase proteins as they are folded by the bacterial Hsp70 system. We observed multiple cycles of chaperone binding and release to an individual client during refolding and determined that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism, whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.

DOI: https://doi.org/10.1126/sciadv.add0922
Nat Commun. 2022 Dec 16. 13(1): 7782

Hepatic neddylation deficiency triggers fatal liver injury via inducing NF-κB-inducing kinase in mice.

Cheng Xu, Hongyi Zhou, Yulan Jin, Khushboo Sahay, Anna Robicsek, Yisong Liu, Kunzhe Dong, Jiliang Zhou, Amanda Barrett, Huabo Su, Weiqin Chen.

The conjugation of neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to target proteins, termed neddylation, participates in many cellular processes and is aberrant in various pathological diseases. Its relevance to liver function and failure remains poorly understood. Herein, we show dysregulated expression of NAE1, a regulatory subunit of the only NEDD8 E1 enzyme, in human acute liver failure. Embryonic- and adult-onset deletion of NAE1 in hepatocytes causes hepatocyte death, inflammation, and fibrosis, culminating in fatal liver injury in mice. Hepatic neddylation deficiency triggers oxidative stress, mitochondrial dysfunction, and hepatocyte reprogramming, potentiating liver injury. Importantly, NF-κB-inducing kinase (NIK), a serine/Thr kinase, is a neddylation substrate. Neddylation of NIK promotes its ubiquitination and degradation. Inhibition of neddylation conversely causes aberrant NIK activation, accentuating hepatocyte damage and inflammation. Administration of N-acetylcysteine, a glutathione surrogate and antioxidant, mitigates liver failure caused by hepatic NAE1 deletion in adult male mice. Therefore, hepatic neddylation is important in maintaining postnatal and adult liver homeostasis, and the identified neddylation targets/pathways provide insights into therapeutically intervening acute liver failure.

DOI: https://doi.org/10.1038/s41467-022-35525-6
J Med Chem. 2022 Dec 14.

Structure-Based Discovery of Selective Histone Deacetylase 8 Degraders with Potent Anticancer Activity.

Jinbo Huang, Jun Zhang, Wenchao Xu, Qiong Wu, Rongsheng Zeng, Zhichao Liu, Wenhui Tao, Qian Chen, Yongqing Wang, Wei-Guo Zhu.

Inducing protein degradation by proteolysis targeting chimeras has gained tremendous momentum as a promising novel therapeutic strategy. Here, we report the design, synthesis, and biological characterization of highly potent proteolysis targeting chimeric small molecules targeting the epigenetic regulator histone deacetylase 8 (HDAC8). We developed potent and effective HDAC8 degraders, as exemplified by SZUH280 (16e), which effectively induced HDAC8 protein degradation and inhibited cancer cell growth even at low micromolar concentrations. Our preliminary mechanistic studies revealed that SZUH280 hampers DNA damage repair in cancer cells, promoting cellular radiosensitization. In mice, a single SZUH280 dose induced rapid and prolonged HDAC8 protein degradation in xenograft tumor tissues. Moreover, SZUH280 alone or in combination with irradiation resulted in long-lasting tumor regression in an A549 tumor mouse model. Our findings qualify a new chemical tool for HDAC8 knockdown and may lead to the development of a new class of cancer therapeutics.

DOI: https://doi.org/10.1021/acs.jmedchem.2c00739
PLoS One. 2022 ;17(12): e0279016

Inositol-requiring enzyme 1α/X-box protein 1 pathway expression is impaired in pediatric cholestatic liver disease explants.

Alyssa Kriegermeier, Angela Hyon, Brian LeCuyer, Susan Hubchak, Xiaoying Liu, Richard M Green.

BACKGROUND: Increased intrahepatic bile acids cause endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is activated to maintain homeostasis. UPR dysregulation, including the inositol-requiring enzyme 1α/X-box protein 1 (IRE1α/XBP1) pathway, is associated with adult liver diseases but has not been characterized in pediatric liver diseases. We evaluated hepatic UPR expression in pediatric cholestatic liver disease (CLD) explants and hypothesize that an inability to appropriately activate the hepatic IRE1α/XBP1 pathway is associated with the pathogenesis of CLD.
METHODS: We evaluated 34 human liver explants, including: pediatric CLD (Alagille, ALGS, and progressive familial intrahepatic cholestasis, PFIC), pediatric non-cholestatic liver disease controls (autoimmune hepatitis, AIH), adult CLD, and normal controls. We performed RNA-seq, quantitative PCR, and western blotting to measure expression differences of the hepatic UPR and other signaling pathways.
RESULTS: Pathway analysis demonstrated that the KEGG 'protein processing in ER' pathway was downregulated in pediatric CLD compared to normal controls. Pediatric CLD had decreased hepatic IRE1α/XBP1 pathway gene expression and decreased protein expression of phosphorylated IRE1α compared to normal controls. IRE1α/XBP1 pathway gene expression was also decreased in pediatric CLD compared to AIH disease controls.
CONCLUSIONS: Pediatric CLD explants have decreased expression of the protective IRE1α/XBP1 pathway and down-regulated KEGG protein processing in the ER pathways. IRE1α/XBP1 pathway expression differences occur when compared to both normal and non-cholestatic disease controls. Attenuated expression of the IRE1α/XBP1 pathway is associated with cholestatic diseases and may be a target for future therapeutics.

DOI: https://doi.org/10.1371/journal.pone.0279016
Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01690-4. [Epub ahead of print]41(11): 111802

Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER.

Hyojeong Hwang, Seongmin Yun, Rachel Braz Arcanjo, Divyanshi, Sijie Chen, Wenyan Mei, Romana A Nowak, Taejoon Kwon, Jing Yang.

  Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development.

Keywords:  CP: Cell biology; CP: Developmental biology; RNA localization; Xenopus; endoplasmic reticulum; maternal RNAs; oocyte maturation

DOI:  https://doi.org/10.1016/j.celrep.2022.111802
iScience. 2022 Dec 22. 25(12): 105659

FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast.

Mayuki Sasaki, Shinichi Nishimura, Yoko Yashiroda, Akihisa Matsuyama, Hideaki Kakeya, Minoru Yoshida.

  FK506-binding protein with a molecular weight of 12 kDa (FKBP12) is a receptor of the immunosuppressive drugs, FK506 and rapamycin. The physiological functions of FKBP12 remain ambiguous because of its nonessentiality and multifunctionality. Here, we show that FKBP12 promotes the utilization of serine as a nitrogen source and regulates the isoleucine biosynthetic pathway in fission yeast. In screening for small molecules that inhibit serine assimilation, we found that the growth of fission yeast cells in medium supplemented with serine as the sole nitrogen source, but not in glutamate-supplemented medium, was suppressed by FKBP12 inhibitors. Knockout of FKBP12 phenocopied the action of these compounds in serine-supplemented medium. Metabolome analyses and genetic screens identified the threonine deaminase, Tda1, to be regulated downstream of FKBP12. Genetic and biochemical analyses unveiled the negative regulation of Tda1 by FKBP12. Our findings reveal new roles of FKBP12 in amino acid biosynthesis and nitrogen metabolism homeostasis.

Keywords:  Biochemistry; Biological sciences; Biosynthesis; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2022.105659
JCI Insight. 2022 11 22. pii: e160606. [Epub ahead of print]7(22):

Small-molecule PROTAC mediates targeted protein degradation to treat STAT3-dependent epithelial cancer.

Jinmei Jin, Yaping Wu, Zeng Zhao, Ye Wu, Yu-Dong Zhou, Sanhong Liu, Qingyan Sun, Guizhu Yang, Jiayi Lin, Dale G Nagle, Jiangjiang Qin, Zhiyuan Zhang, Hong-Zhuan Chen, Weidong Zhang, Shuyang Sun, Xin Luan.

  The aberrant activation of STAT3 is associated with the etiology and progression in a variety of malignant epithelial-derived tumors, including head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). Due to the lack of an enzymatic catalytic site or a ligand-binding pocket, there are no small-molecule inhibitors directly targeting STAT3 that have been approved for clinical translation. Emerging proteolysis targeting chimeric (PROTAC) technology-based approach represents a potential strategy to overcome the limitations of conventional inhibitors and inhibit activation of STAT3 and downstream genes. In this study, the heterobifunctional small-molecule-based PROTACs are successfully prepared from toosendanin (TSN), with 1 portion binding to STAT3 and the other portion binding to an E3 ubiquitin ligase. The optimized lead PROTAC (TSM-1) exhibits superior selectivity, potency, and robust antitumor effects in STAT3-dependent HNSCC and CRC - especially in clinically relevant patient-derived xenografts (PDX) and patient-derived organoids (PDO). The following mechanistic investigation identifies the reduced expression of critical downstream STAT3 effectors, through which TSM-1 promotes cell cycle arrest and apoptosis in tumor cells. These findings provide the first demonstration to our knowledge of a successful PROTAC-targeting strategy in STAT3-dependent epithelial cancer.

Keywords:  Colorectal cancer; Drug therapy; Head and neck cancer; Oncology; Therapeutics

DOI:  https://doi.org/10.1172/jci.insight.160606
JCI Insight. 2022 11 22. pii: e154124. [Epub ahead of print]7(22):

Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis.

Eoin D O'Sullivan, Katie J Mylonas, Rachel Bell, Cyril Carvalho, David P Baird, Carolynn Cairns, Kevin M Gallagher, Ross Campbell, Marie Docherty, Alexander Laird, Neil C Henderson, Tamir Chandra, Kristina Kirschner, Bryan Conway, Gry H Dihazi, Michael Zeisberg, Jeremy Hughes, Laura Denby, Hassan Dihazi, David A Ferenbach.

  Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with aging and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using 2 murine models of injury and repair, we show that obstructive injury generated senescent epithelia, which persisted after resolution of the original injury, promoted ongoing fibrosis, and impeded adaptive repair. Depletion of senescent cells with ABT-263 reduced fibrosis in reversed ureteric obstruction and after renal ischemia/reperfusion injury. We validated these findings in humans, showing that senescence and fibrosis persisted after relieved renal obstruction. We next characterized senescent epithelia in murine renal injury using single-cell RNA-Seq. We extended our classification to human kidney and liver disease and identified conserved profibrotic proteins, which we validated in vitro and in human disease. We demonstrated that increased levels of protein disulfide isomerase family A member 3 (PDIA3) augmented TGF-β-mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduced kidney fibrosis during ongoing renal injury and as such represented a new potential therapeutic pathway. Analysis of the signaling pathways of senescent epithelia connected senescence to organ fibrosis, permitting rational design of antifibrotic therapies.

Keywords:  Bioinformatics; Cell Biology; Cellular senescence; Fibrosis; Nephrology

DOI:  https://doi.org/10.1172/jci.insight.154124