bims-proteo 2024-01-14 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024‒01‒14
thirty papers selected by
Eric Chevet, INSERM

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE.
Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation.
Unveiling the Intercompartmental Signaling Axis: Mitochondrial to ER Stress Response (MERSR) and its Impact on Proteostasis.
Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control.
Journey of Von Hippel-Lindau (VHL) E3 ligase in PROTACs design: From VHL ligands to VHL-based degraders.
Targeted Protein Degradation through Recruitment of the CUL4 Complex Adaptor Protein DDB1.
COP9 signalosome-mediated deneddylation of CULLIN1 is necessary for SCFEBF1 assembly in Arabidopsis thaliana.
Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response.
eIF5A controls mitoprotein import by relieving ribosome stalling at the TIM50 translocase mRNA.
Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis.
Atg8 family proteins, LIR/AIM motifs and other interaction modes.
Identification of TMEM126A as OXA1L-interacting protein reveals cotranslational quality control in mitochondria.
Upregulated pexophagy limits the capacity of selective autophagy.
Substrate identification and specificity profiling of deubiquitylases against endogenously-generated ubiquitin-protein conjugates.
Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism.
Chromatin targeting of the RNF12/RLIM E3 ubiquitin ligase controls transcriptional responses.
Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy.
Response to Bruton's tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy.
The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.
A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.
Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion.
HDAC6/aggresome processing pathway importance for inflammasome formation is context dependent.
Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signaling pathway.
TRIM56 protects against non-alcoholic fatty liver disease via promoting the degradation of fatty acid synthase.
Multi-monoubiquitination controls VASP-mediated actin dynamics.
White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells.
Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes.
Discovery of a PROTAC degrader for METTL3-METTL14 complex.
Protocol for detecting lncRNA-protein interactions in vitro by tRSA RNA pull-down assay.
Attenuating ABHD17 enhances S- palmitoylation, membrane localization and signal transduction of NOD2 and Crohn's disease-associated variants.

Nat Commun. 2024 Jan 09. 15(1): 410

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE.

J Josephine Botsch, Roswitha Junker, Michèle Sorgenfrei, Patricia P Ogger, Luca Stier, Susanne von Gronau, Peter J Murray, Markus A Seeger, Brenda A Schulman, Bastian Bräuning.

Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of Doa10/MARCH6 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, and gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Here, by assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, we reveal crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected Doa10/MARCH6 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity.

DOI: https://doi.org/10.1038/s41467-023-44670-5
bioRxiv. 2023 Dec 20. pii: 2023.12.20.572403. [Epub ahead of print]

Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation.

Hao Li, Zhejian Ji, Joao A Paulo, Steven P Gygi, Tom A Rapoport.

Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex, rather than substrate recruitment. In vivo experiments confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.

DOI: https://doi.org/10.1101/2023.12.20.572403
bioRxiv. 2023 Dec 21. pii: 2023.09.07.556674. [Epub ahead of print]

Unveiling the Intercompartmental Signaling Axis: Mitochondrial to ER Stress Response (MERSR) and its Impact on Proteostasis.

Jeson J Li, Nan Xin, Chunxia Yang, Larissa A Tavizon, Ruth Hong, Travis I Moore, Rebecca George Tharyan, Adam Antebi, Hyun-Eui Kim.

Maintaining protein homeostasis is essential for cellular health. During times of proteotoxic stress, cells deploy unique defense mechanisms to achieve resolution. Our previous research uncovered a cross-compartmental Mitochondrial to Cytosolic Stress Response (MCSR), a unique stress response activated by the perturbation of mitochondrial proteostasis, which ultimately results in the improvement of proteostasis in the cytosol. Here, we found that this signaling axis also influences the unfolded protein response of the endoplasmic reticulum (UPR ER ), suggesting the presence of a Mitochondria to ER Stress Response (MERSR). During MERSR, the IRE1 branch of UPR ER is inhibited, introducing a previously unknown regulatory component of MCSR. Moreover, proteostasis is enhanced through the upregulation of the PERK-eIF2a signaling pathway, increasing phosphorylation of eIF2a and improving the ER's capacity to manage greater proteostasis load. MERSR activation in both poly-glutamine (poly-Q) and amyloid-beta (Aβ) C. elegans disease models also led to improvement in both aggregate burden and overall disease outcome. These findings shed light on the coordination between the mitochondria and the ER in maintaining cellular proteostasis and provides further evidence for the importance of intercompartmental signaling.

DOI: https://doi.org/10.1101/2023.09.07.556674
Mol Cell. 2023 Dec 27. pii: S1097-2765(23)01021-3. [Epub ahead of print]

Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control.

Maxim S Svetlov, Clémence F Dunand, Jose A Nakamoto, Gemma C Atkinson, Haaris A Safdari, Daniel N Wilson, Nora Vázquez-Laslop, Alexander S Mankin.

  Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.

Keywords:  alanine tailing; antibiotics; nascent peptide; protein folding; protein synthesis; ribosome rescue; translation

DOI:  https://doi.org/10.1016/j.molcel.2023.12.002
Eur J Med Chem. 2023 Dec 14. pii: S0223-5234(23)01008-5. [Epub ahead of print]265 116041

Journey of Von Hippel-Lindau (VHL) E3 ligase in PROTACs design: From VHL ligands to VHL-based degraders.

Nisha Setia, Haider Thaer Abdulhameed Almuqdadi, Mohammad Abid.

  The scientific community has shown considerable interest in proteolysis-targeting chimeras (PROTACs) in the last decade, indicating their remarkable potential as a means of achieving targeted protein degradation (TPD). Not only are PROTACs seen as valuable tools in molecular biology but their emergence as a modality for drug discovery has also garnered significant attention. PROTACs bind to E3 ligases and target proteins through respective ligands connected via a linker to induce proteasome-mediated protein degradation. The discovery of small molecule ligands for E3 ligases has led to the prevalent use of various E3 ligases in PROTAC design. Furthermore, the incorporation of different types of linkers has proven beneficial in enhancing the efficacy of PROTACs. By far more than 3300 PROTACs have been reported in the literature. Notably, Von Hippel-Lindau (VHL)-based PROTACs have surfaced as a propitious strategy for targeting proteins, even encompassing those that were previously considered non-druggable. VHL is extensively utilized as an E3 ligase in the advancement of PROTACs owing to its widespread expression in various tissues and well-documented binders. Here, we review the discovery of VHL ligands, the types of linkers employed to develop VHL-based PROTACs, and their subsequent modulation to design advanced non-conventional degraders to target various disease-causing proteins. Furthermore, we provide an overview of other E3 ligases recruited in the field of PROTAC technology.

Keywords:  E3 ligase; Ligands; PROTACs; Protein degradation; Ubiquitin proteasome system; Von hippel-lindau (VHL)

DOI:  https://doi.org/10.1016/j.ejmech.2023.116041
ACS Chem Biol. 2024 Jan 08.

Targeted Protein Degradation through Recruitment of the CUL4 Complex Adaptor Protein DDB1.

Margot Meyers, Sabine Cismoski, Anoohya Panidapu, Barbara Chie-Leon, Daniel K Nomura.

Targeted protein degradation has arisen as a powerful therapeutic modality for eliminating proteins. Thus far, most heterobifunctional proteolysis targeting chimeras (PROTACs) have utilized recruiters against substrate receptors of Cullin RING E3 ubiquitin ligases, such as cereblon and VHL. However, previous studies have surprisingly uncovered molecular glue degraders that exploit a CUL4 adaptor protein DDB1 to degrade neosubstrate proteins. Here, we sought to investigate whether DDB1 recruiters can be discovered that can be exploited for PROTAC applications. We utilized activity-based protein profiling and cysteine chemoproteomic screening to identify a covalent recruiter that targets C173 on DDB1 and exploited this recruiter to develop PROTACs against BRD4 and androgen receptor (AR). We demonstrated that the BRD4 PROTAC results in selective degradation of the short BRD4 isoform over the long isoform in a proteasome, NEDDylation, and DDB1-dependent manner. We also demonstrated degradation of AR with the AR PROTAC in prostate cancer cells. Our study demonstrated that covalent chemoproteomic approaches can be used to discover recruiters against Cullin RING adapter proteins and that these recruiters can be used for PROTAC applications to degrade neo-substrates.

DOI: https://doi.org/10.1021/acschembio.3c00487
Cell Rep. 2024 Jan 06. pii: S2211-1247(23)01649-2. [Epub ahead of print]43(1): 113638

COP9 signalosome-mediated deneddylation of CULLIN1 is necessary for SCFEBF1 assembly in Arabidopsis thaliana.

Jie Dong, Yuanyuan Li, Shuyang Cheng, Xuehui Li, Ning Wei.

  Functions of the SKP1-CUL1-F box (SCF) ubiquitin E3 ligases are essential in plants. The F box proteins (FBPs) are substrate receptors that recruit substrates and assemble an active SCF complex, but the regulatory mechanism underlying the FBPs binding to CUL1 to activate the SCF cycle is not fully understood. We show that Arabidopsis csn1-10 is defective in SCFEBF1-mediated PIF3 degradation during de-etiolation, due to impaired association of EBF1 with CUL1 in csn1-10. EBF1 preferentially associates with un-neddylated CUL1 that is deficient in csn1-10 and the EBF1-CUL1 binding is rescued by the neddylation inhibitor MLN4924. Furthermore, we identify a subset of FBPs with impaired binding to CUL1 in csn1-10, indicating their assembly to form SCF complexes may depend on COP9 signalosome (CSN)-mediated deneddylation of CUL1. This study reports that a key role of CSN-mediated CULLIN deneddylation is to gate the binding of the FBP-substrate module to CUL1, thus initiating the SCF cycle of substrate ubiquitination.

Keywords:  Arabidopsis thaliana; COP9 signalosome; CP: Molecular biology; CP: Plants; CUL1; EBF1; F-box proteins; PIF3; SCF complex; neddylation; protein degradation; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2023.113638
Redox Biol. 2024 Jan 05. pii: S2213-2317(24)00001-6. [Epub ahead of print]69 103025

Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response.

Mingzhan Xue, Zehra Irshad, Naila Rabbani, Paul J Thornalley.

  The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.

Keywords:  ER stress; Glycation; Glycemic disease; Hyperglycemia; Methylglyoxal; Unfolded protein response

DOI:  https://doi.org/10.1016/j.redox.2024.103025
bioRxiv. 2023 Dec 19. pii: 2023.12.19.572290. [Epub ahead of print]

eIF5A controls mitoprotein import by relieving ribosome stalling at the TIM50 translocase mRNA.

Marina Barba-Aliaga, Vanessa Bernal, Cynthia Rong, Brian M Zid, Paula Alepuz.

The efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A is primarily known as an elongation factor which binds ribosomes to alleviate ribosome stalling at sequences encoding polyprolines or combinations of proline with glycine and charged amino acids. eIF5A is known to impact the mitochondrial function across a variety of species although the precise molecular mechanism underlying this impact remains unclear. We found that depletion of eIF5A in yeast drives reduced translation and levels of TCA cycle and oxidative phosphorylation proteins. We further found that loss of eIF5A leads to the accumulation of mitoprotein precursors in the cytosol as well as to the induction of a mitochondrial import stress response. Here we identify an essential polyproline-containing protein as a direct eIF5A target for translation: the mitochondrial inner membrane protein Tim50, which is the receptor sub-unit of the TIM23 translocase complex. We show how eIF5A directly controls mitochondrial protein import through the alleviation of ribosome stalling along TIM50 mRNA at the mitochondrial surface. Removal of the polyprolines from Tim50 rescues the mitochondrial import stress response, as well as the translation of oxidative phosphorylation reporter genes in an eIF5A loss of function. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by reducing ribosome stalling and facilitating protein translation, thereby positively impacting the mitochondrial import process.

DOI: https://doi.org/10.1101/2023.12.19.572290
EMBO J. 2024 Jan 11.

Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis.

Ching-Fang Chang, Amanda L Gunawan, Irene Liparulo, Peter-James H Zushin, Kaitlyn Vitangcol, Greg A Timblin, Kaoru Saijo, Biao Wang, Güneş Parlakgül, Ana Paula Arruda, Andreas Stahl.

  Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.

Keywords:  Brown Adipose Tissue; Coenzyme Q; FGF21; Mitochondrial Unfolded Protein Response; Mitohormesis

DOI:  https://doi.org/10.1038/s44318-023-00008-x
Autophagy Rep. 2023 Dec 31. pii: 27694127.2023.2188523. [Epub ahead of print]2(1):

Atg8 family proteins, LIR/AIM motifs and other interaction modes.

Vladimir V Rogov, Ioannis P Nezis, Panagiotis Tsapras, Hong Zhang, Yasin Dagdas, Nobuo N Noda, Hitoshi Nakatogawa, Martina Wirth, Stephane Mouilleron, David G McEwan, Christian Behrends, Vojo Deretic, Zvulun Elazar, Sharon A Tooze, Ivan Dikic, Trond Lamark, Terje Johansen.

  The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.

Keywords:  AIM; Atg8; Autophagy; LDS; LIR; UDS; phosphorylation; protein-protein interaction

DOI:  https://doi.org/10.1080/27694127.2023.2188523
Mol Cell. 2023 Dec 29. pii: S1097-2765(23)01031-6. [Epub ahead of print]

Identification of TMEM126A as OXA1L-interacting protein reveals cotranslational quality control in mitochondria.

Sabine Poerschke, Silke Oeljeklaus, Luis Daniel Cruz-Zaragoza, Alexander Schenzielorz, Drishan Dahal, Hauke Sven Hillen, Hirak Das, Laura Sophie Kremer, Anusha Valpadashi, Mirjam Breuer, Johannes Sattmann, Ricarda Richter-Dennerlein, Bettina Warscheid, Sven Dennerlein, Peter Rehling.

  Cellular proteostasis requires transport of polypeptides across membranes. Although defective transport processes trigger cytosolic rescue and quality control mechanisms that clear translocases and membranes from unproductive cargo, proteins that are synthesized within mitochondria are not accessible to these mechanisms. Mitochondrial-encoded proteins are inserted cotranslationally into the inner membrane by the conserved insertase OXA1L. Here, we identify TMEM126A as a OXA1L-interacting protein. TMEM126A associates with mitochondrial ribosomes and translation products. Loss of TMEM126A leads to the destabilization of mitochondrial translation products, triggering an inner membrane quality control process, in which newly synthesized proteins are degraded by the mitochondrial iAAA protease. Our data reveal that TMEM126A cooperates with OXA1L in protein insertion into the membrane. Upon loss of TMEM126A, the cargo-blocked OXA1L insertase complexes undergo proteolytic clearance by the iAAA protease machinery together with its cargo.

Keywords:  mitochondria; mitochondrial quality control; mitochondrial translation

DOI:  https://doi.org/10.1016/j.molcel.2023.12.013
Nat Commun. 2024 Jan 09. 15(1): 375

Upregulated pexophagy limits the capacity of selective autophagy.

Kyla Germain, Raphaella W L So, Laura F DiGiovanni, Joel C Watts, Robert H J Bandsma, Peter K Kim.

Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson's Disease and Huntington's Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.

DOI: https://doi.org/10.1038/s41467-023-44005-4
bioRxiv. 2023 Dec 20. pii: 2023.12.20.572581. [Epub ahead of print]

Substrate identification and specificity profiling of deubiquitylases against endogenously-generated ubiquitin-protein conjugates.

Valentina Rossio, Joao A Paulo, Xinyue Liu, Steven P Gygi, Randall W King.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high impact DUBs (USP7, USP9X, USP36, USP15 and USP24) that each reduced ubiquitylation of over ten percent of the isolated proteins. Candidate substrates of high impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

DOI: https://doi.org/10.1101/2023.12.20.572581
Nature. 2024 Jan 10.

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism.

Daniel L Dunkelmann, Carlos Piedrafita, Alexandre Dickson, Kim C Liu, Thomas S Elliott, Marc Fiedler, Dom Bellini, Andrew Zhou, Daniele Cervettini, Jason W Chin.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several β-amino acids, α,α-disubstituted-amino acids and β-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of β-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

DOI: https://doi.org/10.1038/s41586-023-06897-6
Life Sci Alliance. 2024 Mar;pii: e202302282. [Epub ahead of print]7(3):

Chromatin targeting of the RNF12/RLIM E3 ubiquitin ligase controls transcriptional responses.

Carmen Espejo-Serrano, Catriona Aitken, Beatrice F Tan, Danielle G May, Rachel J Chrisopulos, Kyle J Roux, Jeroen Aa Demmers, Samuel G Mackintosh, Joost Gribnau, Francisco Bustos, Cristina Gontan, Greg M Findlay.

Protein ubiquitylation regulates key biological processes including transcription. This is exemplified by the E3 ubiquitin ligase RNF12/RLIM, which controls developmental gene expression by ubiquitylating the REX1 transcription factor and is mutated in an X-linked intellectual disability disorder. However, the precise mechanisms by which ubiquitylation drives specific transcriptional responses are not known. Here, we show that RNF12 is recruited to specific genomic locations via a consensus sequence motif, which enables co-localisation with REX1 substrate at gene promoters. Surprisingly, RNF12 chromatin recruitment is achieved via a non-catalytic basic region and comprises a previously unappreciated N-terminal autoinhibitory mechanism. Furthermore, RNF12 chromatin targeting is critical for REX1 ubiquitylation and downstream RNF12-dependent gene regulation. Our results demonstrate a key role for chromatin in regulation of the RNF12-REX1 axis and provide insight into mechanisms by which protein ubiquitylation enables programming of gene expression.

DOI: https://doi.org/10.26508/lsa.202302282
Bio Protoc. 2024 Jan 05. 14(1): e4917

Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy.

Wenxin Zhang, Taki Nishimura, Sharon A Tooze.

  Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome. The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion. They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation. In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12-ATG5-ATG16L1. However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction. In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8. This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation. It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation. Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout. Key features • Preparation of ATG7 E1 from insect cells (Sf9 cells). • Preparation of ATG3 E2 from bacteria (E. coli). • Preparation of LC3B S3C from bacteria (E. coli). • Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.

Keywords:  Autophagy; Fluorescence spectroscopy; In vitro ATG8 lipidation; Liposomes; NBD; Real-time ATG8 lipidation assay; Site-directed fluorescence; Ubiquitin-like conjugation

DOI:  https://doi.org/10.21769/BioProtoc.4917
Cancer Cell. 2024 Jan 06. pii: S1535-6108(23)00447-6. [Epub ahead of print]

Response to Bruton's tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy.

James D Phelan, Sebastian Scheich, Jaewoo Choi, George W Wright, Björn Häupl, Ryan M Young, Sara A Rieke, Martine Pape, Yanlong Ji, Henning Urlaub, Arnold Bolomsky, Carmen Doebele, Alena Zindel, Tanja Wotapek, Monica Kasbekar, Brett Collinge, Da Wei Huang, Zana A Coulibaly, Vivian M Morris, Xiaoxuan Zhuang, Julius C Enssle, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Zhuo Wang, Andy D Tran, Christopher J Shoemaker, Galina Shevchenko, Daniel J Hodson, Arthur L Shaffer, Louis M Staudt, Thomas Oellerich.

  Diffuse large B cell lymphoma (DLBCL) is an aggressive, profoundly heterogeneous cancer, presenting a challenge for precision medicine. Bruton's tyrosine kinase (BTK) inhibitors block B cell receptor (BCR) signaling and are particularly effective in certain molecular subtypes of DLBCL that rely on chronic active BCR signaling to promote oncogenic NF-κB. The MCD genetic subtype, which often acquires mutations in the BCR subunit, CD79B, and in the innate immune adapter, MYD88L265P, typically resists chemotherapy but responds exceptionally to BTK inhibitors. However, the underlying mechanisms of response to BTK inhibitors are poorly understood. Herein, we find a non-canonical form of chronic selective autophagy in MCD DLBCL that targets ubiquitinated MYD88L265P for degradation in a TBK1-dependent manner. MCD tumors acquire genetic and epigenetic alterations that attenuate this autophagic tumor suppressive pathway. In contrast, BTK inhibitors promote autophagic degradation of MYD88L265P, thus explaining their exceptional clinical benefit in MCD DLBCL.

Keywords:  Bruton’s tyrosine kinase; DLBCL; autophagy; functional genomics; proteomics; targeted therapy

DOI:  https://doi.org/10.1016/j.ccell.2023.12.019
Cell Rep. 2024 Jan 05. pii: S2211-1247(23)01642-X. [Epub ahead of print]43(1): 113631

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.

Clément Maghe, Kilian Trillet, Gwennan André-Grégoire, Mathilde Kerhervé, Laura Merlet, Kathryn A Jacobs, Kristine Schauer, Nicolas Bidère, Julie Gavard.

  Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

Keywords:  CP: Cancer; CP: Cell biology; Niemann-Pick disease; SREBP2; autophagy; cholesterol; glioblastoma; glioma; late endosome; lysosome; proteome; transporter

DOI:  https://doi.org/10.1016/j.celrep.2023.113631
Nat Biotechnol. 2024 Jan 08.

A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.

Cory A Ocasio, Marc P Baggelaar, James Sipthorp, Ana Losada de la Lastra, Manuel Tavares, Jana Volarić, Christelle Soudy, Elisabeth M Storck, Jack W Houghton, Susana A Palma-Duran, James I MacRae, Goran Tomić, Lotte Carr, Julian Downward, Ulrike S Eggert, Edward W Tate.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

DOI: https://doi.org/10.1038/s41587-023-02030-0
Mol Metab. 2024 Jan 09. pii: S2212-8778(24)00005-X. [Epub ahead of print] 101874

Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion.

Zhouji Chen, Shiyu Wang, Anita Pottekat, Alec Duffey, Insook Jang, Benny H Chang, Jaehyung Cho, Brian N Finck, Nicholas O Davidson, Randal J Kaufman.

  The assembly and secretion of hepatic very low-density lipoprotein (VLDL) plays pivotal roles in hepatic and plasma lipid hemostasis and the development of fatty liver disease. Protein disulfide isomerase A1 (PDIA1/P4HB) functions as a molecular chaperone and an oxidoreductase that is essential for protein folding in the endoplasmic reticulum. While PDIA1 biochemical functions have been extensively investigated in vitro, little is known about its physiological requirement in vivo. PDIA1 is a subunit of the microsomal triglyceride transfer protein (MTTP) complex that is essential for VLDL biosynthesis, a pivotal process that regulates liver and whole-body lipid homeostasis. Here, we find that hepatocyte-specific deletion of P4hb (Pdia1-LKO) disrupts MTTP function and VLDL production, leading to hepatic steatosis and severe hypolipidemia. Surprisingly, Pdia1-deletion did not affect mRNA expression or protein stability of MTTP but prevented Mttp mRNA translation. Importantly, besides its essential role in MTTP synthesis and function, we also found that PDIA1 interacts with APOB in an MTTP-independent manner via its molecular chaperone function to supports APOB folding and secretion. Together, our results demonstrate that PDIA1 plays indispensable roles in both APOB folding and MTTP synthesis and activity to support VLDL secretion and regulate liver and plasma lipid homeostasis.

Keywords:  VLDL secretion; fatty liver disease; lipoprotein metabolism; microsomal triglyceride transfer protein; protein disulfide isomerase A1; protein folding; unfolded protein response

DOI:  https://doi.org/10.1016/j.molmet.2024.101874
J Biol Chem. 2024 Jan 08. pii: S0021-9258(24)00014-0. [Epub ahead of print] 105638

HDAC6/aggresome processing pathway importance for inflammasome formation is context dependent.

Longlong Wang, Shihua Shi, Adeline Unterreiner, Ronan Kapetanovic, Sucheta Ghosh, Jacint Sanchez, Selma Aslani, Yuan Xiong, Chi-Lin Hsu, Katherine A Donovan, Christopher J Farady, Eric S Fischer, Frédéric Bornancin, Patrick Matthias.

  The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1β and IL-18. Previous research has shown that in immortalized bone marrow-derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocytes cell lines expressing a synthetic protein blocking the HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected. Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context dependent.

Keywords:  DARPin; HDAC6; Inflammasome; degrader; inhibitors; interleukin

DOI:  https://doi.org/10.1016/j.jbc.2024.105638
Mol Cell. 2024 Jan 01. pii: S1097-2765(23)01032-8. [Epub ahead of print]

Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signaling pathway.

Iqbal Dulloo, Michael Tellier, Clémence Levet, Anissa Chikh, Boyan Zhang, Diana C Blaydon, Catherine M Webb, David P Kelsell, Matthew Freeman.

  iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signaling; they function primarily by recognizing transmembrane domains of their clients. Here, we report a mechanistically distinct nuclear function of iRhoms, showing that both human and mouse iRhom2 are non-canonical substrates of signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the transcriptome, in part by binding C-terminal binding proteins (CtBPs). The biological significance of nuclear iRhom2 is indicated by elevated levels in skin biopsies of patients with psoriasis, tylosis with oesophageal cancer (TOC), and non-epidermolytic palmoplantar keratoderma (NEPPK); increased iRhom2 cleavage in a keratinocyte model of psoriasis; and nuclear iRhom2 promoting proliferation of keratinocytes. Overall, this work identifies an unexpected SPC-dependent ER-to-nucleus signaling pathway and demonstrates that iRhoms can mediate nuclear signaling.

Keywords:  CtBP1; CtBP2; RHBDF1; RHBDF2; RNA-seq; TOC; psoriasis; rhomboid; skin

DOI:  https://doi.org/10.1016/j.molcel.2023.12.012
J Clin Invest. 2024 Jan 11. pii: e166149. [Epub ahead of print]

TRIM56 protects against non-alcoholic fatty liver disease via promoting the degradation of fatty acid synthase.

Suowen Xu, Xiumei Wu, Sichen Wang, Mengyun Xu, Tingyu Fang, Xiaoxuan Ma, Meijie Chen, Jiajun Fu, Juan Guo, Song Tian, Tian Tian, Xu Cheng, Hailong Yang, Junjie Zhou, Zhenya Wang, Yanjun Yin, Wen Xu, Fen Xu, Jinhua Yan, Zhihua Wang, Sihui Luo, Xiao-Jing Zhang, Yan-Xiao Ji, Jianping Weng.

  Nonalcoholic liver disease (NAFLD) encompasses a disease continuum from simple steatosis, to non-alcoholic steatohepatitis (NASH). However, there are currently no approved pharmacotherapies for NAFLD although several drugs are in advanced stages of clinical development. Because of the complex pathophysiology and heterogeneity of NALFD, identification of potential therapeutic targets is clinically important. Here, we demonstrated that TRIM56 protein abundance is markedly downregulated in the livers of individuals with NAFLD and mice fed a high-fat diet. Hepatocyte-specific ablation of TRIM56 exacerbated the progression of NAFLD, while hepatic TRIM56 overexpression suppressed it. Integrative analyses of interactomic and transcriptomic profiling revealed a pivotal role of TRIM56 in lipid metabolism and identified lipogenesis factor FASN as a direct binding partner of TRIM56. TRIM56 directly interacts with FASN and triggers its K48-linked ubiquitination-dependent degradation. Finally, by using AI-based virtual screening, we discovered an orally bioavailable small-molecule inhibitor of FASN (named FASstatin) which potentiates TRIM56-mediated FASN ubiquitination. Therapeutic administration of FASstatin improved NAFLD and NASH pathologies in mice with optimal safety, tolerability and pharmacokinetic profile. Our findings provide the proof-of-concept that targeting the TRIM56/FASN axis in hepatocytes may offer potential therapeutic avenues to treat NAFLD.

Keywords:  Hepatology; Ubiquitin-proteosome system

DOI:  https://doi.org/10.1172/JCI166149
J Cell Sci. 2024 Jan 12. pii: jcs.261527. [Epub ahead of print]

Multi-monoubiquitination controls VASP-mediated actin dynamics.

Laura E McCormick, Cristian Suarez, Laura E Herring, Kevin S Cannon, David R Kovar, Nicholas G Brown, Stephanie L Gupton.

  The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitination regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitination impacts VASP activity was unknown. Here we show that mimicking multi-monoubiquitination of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitinated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitinated VASP maintained the ability to bind and protect barbed ends from capping protein. Lastly, we demonstrate the electroporation of recombinant multi-monoubiquitinated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitination controls VASP-mediated actin dynamics.

Keywords:  Actin; Ubiquitination; VASP

DOI:  https://doi.org/10.1242/jcs.261527
bioRxiv. 2024 Jan 02. pii: 2023.12.18.566359. [Epub ahead of print]

White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells.

Sunny Hoi-Sang Yeung, Ralph Hon-Sun Lee, Gerald Wai-Yeung Cheng, Iris Wai-Ting Ma, Julia Kofler, Candice Kent, Fulin Ma, Karl Herrup, Myriam Fornage, Ken Arai, Kai-Hei Tse.

White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. GWAS identified TRIM47 at 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found selectively expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially-induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we identified the highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription and vacuole formation. Together, we confirm that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation.SUMMARY STATEMENT: TRIM47, top genetic risk factor for white matter hyperintensity formation, is a negative regulator of autophagy in brain endothelial cells and implicates a novel cellular mechanism for age-related cerebrovascular changes.

DOI: https://doi.org/10.1101/2023.12.18.566359
Mol Biol Cell. 2024 Jan 10. mbcE23080328

Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes.

Jan van der Beek, Cilia de Heus, Paolo Sanza, Nalan Liv, Judith Klumperman.

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These 'HOPS bodies' were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.

DOI: https://doi.org/10.1091/mbc.E23-08-0328
Cell Chem Biol. 2024 Jan 02. pii: S2451-9456(23)00439-7. [Epub ahead of print]

Discovery of a PROTAC degrader for METTL3-METTL14 complex.

Wenhao Du, Yuting Huang, Xiaoai Chen, Yue Deng, Yaoliang Sun, Hong Yang, Qiongyu Shi, Feifei Wu, Guobin Liu, He Huang, Jian Ding, Xun Huang, Shilin Xu.

  N6-methyladenosine (m6A) methylation is the most abundant type of RNA modification that is mainly catalyzed by the METTL3-METTL14 methyltransferase complex. This complex has been linked to multiple cancers and is considered a promising therapeutic target for acute myeloid leukemia (AML). However, only a few METTL3 inhibitors targeting the catalytic activity were developed recently. Here, we present the discovery of WD6305 as the potent and selective proteolysis-targeting chimera (PROTAC) degrader of METTL3-METTL14 complex. WD6305 suppresses m6A modification and the proliferation of AML cells, and promotes apoptosis much more effectively than its parent inhibitor. WD6305 also affects a variety of signaling pathways related to the development and proliferation of AML. Collectively, our study reveals PROTAC degradation of METTL3-METTL14 complex as a potential anti-leukemic strategy and provides desirable chemical tool for further understanding METTL3-METTL14 protein functions.

Keywords:  Degrader; METTL3; Methyltransferase; PROTAC; m(6)A

DOI:  https://doi.org/10.1016/j.chembiol.2023.12.009
STAR Protoc. 2024 Jan 05. pii: S2666-1667(23)00785-2. [Epub ahead of print]5(1): 102818

Protocol for detecting lncRNA-protein interactions in vitro by tRSA RNA pull-down assay.

Liyun Jiang, Jun Yang, Reqing He, Youlin Zhu, Dong Wang.

  Long non-coding RNAs (lncRNAs) work together with diverse RNA-binding proteins (RBPs) to fulfill key regulations in important cellular functions. Here, we present a protocol to detect lncRNA-RBP interactions in vitro using a tRNA scaffold containing a streptavidin aptamer pull-down assay. We describe steps for preparing both protein and lncRNA transcripts, lncRNA-protein interaction detection with an in vitro binding assay, and western blot analysis. This protocol is applicable to screen for RNA-interacting proteins using cell lysates followed by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Yang et al. (2023).1.

Keywords:  Gene Expression; Molecular Biology; Plant sciences

DOI:  https://doi.org/10.1016/j.xpro.2023.102818
bioRxiv. 2023 Dec 21. pii: 2023.12.20.572362. [Epub ahead of print]

Attenuating ABHD17 enhances S- palmitoylation, membrane localization and signal transduction of NOD2 and Crohn's disease-associated variants.

Charneal L Dixon, Noah R Martin, Micah J Niphakis, Benjamin F Cravatt, Gregory D Fairn.

NOD2 is an intracellular innate immune receptor that senses bacterial peptidoglycans. Although soluble in the cytosol, a portion of the protein is associated with the plasma membrane and endosomal compartments for microbial surveillance. Palmitoylation of NOD2 by zDHHC5 promotes its membrane recruitment to drive proinflammatory and antimicrobial responses to pathogenic invasion. A depalmitoylation step by an unknown protein, thioesterase, releases NOD2 from membranes into the cytosol, where the protein can then enter a new cycle of palmitoylation-depalmitoylation. Here, we identify α/β -hydrolase domain-containing protein 17 isoforms (ABHD17A, 17B, 17C) as the thioesterases responsible for depalmitoylation of NOD2. Inhibiting ABHD17 increased the plasmalemmal localization of both wild-type NOD2 and a subset of hypo-palmitoylated Crohn's disease-associated variants, resulting in increased NF-κB activation and production of pro-inflammatory cytokines in epithelial cells. These results suggest that targeted inhibition of ABHD17 may rescue some Crohn's disease-associated NOD2 variants.

DOI: https://doi.org/10.1101/2023.12.20.572362