bims-proteo 2024-06-30 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024‒06‒30
27 papers selected by
Eric Chevet, INSERM

Alkylamine-tethered molecules recruit FBXO22 for targeted protein degradation.
Mechanism of chaperone coordination during cotranslational protein folding in bacteria.
Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD.
Nub1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome.
Stress-dependent condensate formation regulated by the ubiquitin-related modifier Urm1.
Stereochemical inversion at a 1,4-cyclohexyl PROTAC linker fine-tunes conformation and binding affinity.
Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.
Gradual ER calcium depletion induces a progressive and reversible UPR signaling.
A metabolic redox relay supports ER proinsulin export in pancreatic islet β-cells.
Preferential secretion of oxidation-sensitive proteins by unconventional pathways: Why is this important for inflammation?
Pathogenic tau induces an adaptive elevation in mRNA translation rate at early stages of disease.
Protein disulfide isomerase is essential for spermatogenesis in mice.
Discovery of Monovalent Direct Degraders of BRD4 That Act Via the Recruitment of DCAF11.
Recruitment of Ahsa1 to Hsp90 is regulated by a conserved peptide that inhibits ATPase stimulation.
Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy.
A dual role of ERGIC-localized Rabs in TMED10-mediated unconventional protein secretion.
Targeting the TRIM14/USP14 Axis Enhances Immunotherapy Efficacy by Inducing Autophagic Degradation of PD-L1.
Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.
SDS22 coordinates the assembly of holoenzymes from nascent protein phosphatase-1.
Microglial-derived C1q integrates into neuronal ribonucleoprotein complexes and impacts protein homeostasis in the aging brain.
Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention.
The extracellular matrix integrates mitochondrial homeostasis.
Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome.
N-terminal cysteine acetylation and oxidation patterns may define protein stability.
The proteogenomic landscape of multiple myeloma reveals insights into disease biology and therapeutic opportunities.
METTL3-mediated chromatin contacts promote stress granule phase separation through metabolic reprogramming during senescence.
Pan-cancer proteogenomics expands the landscape of therapeutic targets.

Nat Commun. 2024 Jun 26. 15(1): 5409

Alkylamine-tethered molecules recruit FBXO22 for targeted protein degradation.

Chrysanthi Kagiou, Jose A Cisneros, Jakob Farnung, Joanna Liwocha, Fabian Offensperger, Kevin Dong, Ka Yang, Gary Tin, Christina S Horstmann, Matthias Hinterndorfer, Joao A Paulo, Natalie S Scholes, Juan Sanchez Avila, Michaela Fellner, Florian Andersch, J Thomas Hannich, Johannes Zuber, Stefan Kubicek, Steven P Gygi, Brenda A Schulman, Georg E Winter.

Targeted protein degradation (TPD) relies on small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation by the proteasome. Only a few of the approximately 600 human E3 ligases are currently amenable to this strategy. This limits the actionable target space and clinical opportunities and thus establishes the necessity to expand to additional ligases. Here we identify and characterize SP3N, a specific degrader of the prolyl isomerase FKBP12. SP3N features a minimal design, where a known FKBP12 ligand is appended with a flexible alkylamine tail that conveys degradation properties. We found that SP3N is a precursor and that the alkylamine is metabolized to an active aldehyde species that recruits the SCFFBXO22 ligase for FKBP12 degradation. Target engagement occurs via covalent adduction of Cys326 in the FBXO22 C-terminal domain, which is critical for ternary complex formation, ubiquitylation and degradation. This mechanism is conserved for two recently reported alkylamine-based degraders of NSD2 and XIAP, thus establishing alkylamine tethering and covalent hijacking of FBXO22 as a generalizable TPD strategy.

DOI: https://doi.org/10.1038/s41467-024-49739-3
Mol Cell. 2024 Jun 18. pii: S1097-2765(24)00480-5. [Epub ahead of print]

Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

Alžběta Roeselová, Sarah L Maslen, Santosh Shivakumaraswamy, Grant A Pellowe, Steven Howell, Dhira Joshi, Joanna Redmond, Svend Kjær, J Mark Skehel, David Balchin.

  Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

Keywords:  DnaJ; DnaK; Hsp40; Hsp70; Trigger factor; cotranslational folding; molecular chaperones; protein folding; ribosomes

DOI:  https://doi.org/10.1016/j.molcel.2024.06.002
Nat Struct Mol Biol. 2024 Jun 25.

Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD.

Elias Adriaenssens, Thanh Ngoc Nguyen, Justyna Sawa-Makarska, Grace Khuu, Martina Schuschnig, Stephen Shoebridge, Marvin Skulsuppaisarn, Emily Maria Watts, Kitti Dora Csalyi, Benjamin Scott Padman, Michael Lazarou, Sascha Martens.

Mitophagy preserves overall mitochondrial fitness by selectively targeting damaged mitochondria for degradation. The regulatory mechanisms that prevent PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin (PINK1/Parkin)-dependent mitophagy and other selective autophagy pathways from overreacting while ensuring swift progression once initiated are largely elusive. Here, we demonstrate how the TBK1 (TANK-binding kinase 1) adaptors NAP1 (NAK-associated protein 1) and SINTBAD (similar to NAP1 TBK1 adaptor) restrict the initiation of OPTN (optineurin)-driven mitophagy by competing with OPTN for TBK1. Conversely, they promote the progression of nuclear dot protein 52 (NDP52)-driven mitophagy by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200. Notably, OPTN emerges as the primary recruiter of TBK1 during mitophagy initiation, which in return boosts NDP52-mediated mitophagy. Our results thus define NAP1 and SINTBAD as cargo receptor rheostats, elevating the threshold for mitophagy initiation by OPTN while promoting the progression of the pathway once set in motion by supporting NDP52. These findings shed light on the cellular strategy to prevent pathway hyperactivity while still ensuring efficient progression.

DOI: https://doi.org/10.1038/s41594-024-01338-y
bioRxiv. 2024 Jun 12. pii: 2024.06.12.598715. [Epub ahead of print]

Nub1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome.

Connor Arkinson, Ken C Dong, Christine L Gee, Shawn M Costello, Susan Marqusee, Andreas Martin.

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor Nub1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system and revealed that Nub1 utilizes FAT10's intrinsic instability to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin- and p97-independent manner. Through hydrogen-deuterium exchange, structural modeling, and site-directed mutagenesis, we identified the formation of a peculiar complex with FAT10 that activates Nub1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic Nub1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These studies thus identified a novel mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.

DOI: https://doi.org/10.1101/2024.06.12.598715
Cell. 2024 Jun 26. pii: S0092-8674(24)00649-4. [Epub ahead of print]

Stress-dependent condensate formation regulated by the ubiquitin-related modifier Urm1.

Lucas V Cairo, Xiaoyu Hong, Martin B D Müller, Patricia Yuste-Checa, Chandhuru Jagadeesan, Andreas Bracher, Sae-Hun Park, Manajit Hayer-Hartl, F Ulrich Hartl.

  The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.

Keywords:  Saccharomyces cerevisiae; biomolecular condensate; heat stress; liquid-liquid phase separation; nucleolus; nucleus; stress granules; uba4; ubiquitin-like modifier; urm1

DOI:  https://doi.org/10.1016/j.cell.2024.06.009
Bioorg Med Chem Lett. 2024 Jun 26. pii: S0960-894X(24)00263-4. [Epub ahead of print] 129861

Stereochemical inversion at a 1,4-cyclohexyl PROTAC linker fine-tunes conformation and binding affinity.

Martina Pierri, Xingui Liu, Alena Kroupova, Zoe Rutter, Alex J Hallatt, Alessio Ciulli.

  Proteolysis targeting chimeras (PROTACs) are heterobifunctional small-molecule degraders made of a linker connecting a target-binding moiety to a ubiquitin E3 ligase-binding moiety. The linker unit is known to influence the physicochemical and pharmacokinetic properties of PROTACs, as well as the properties of ternary complexes, in turn impacting on their degradation activity in cells and in vivo. Our LRRK2 PROTAC XL01126, bearing a trans-cyclohexyl group in the linker, is a better and more cooperative degrader than its corresponding cis- analogue despite its much weaker binary binding affinities. Here, we investigate how this subtle stereocenter alteration in the linker affects the ligand binding affinity to the E3 ligase VHL. We designed a series of molecular matched pairs, truncating from the full PROTACs down to the VHL ligand, and find that across the series the trans-cyclohexyl compounds showed consistently weaker VHL-binding affinity compared to the cis- counterparts. High-resolution co-crystal structures revealed that the trans linker exhibits a rigid stick-out conformation, while the cis linker collapses into a folded-back conformation featuring a network of intramolecular contacts and long-range interactions with VHL. These observations are noteworthy as they reveal how a single stereochemical inversion within a PROTAC linker impacts conformational rigidity and binding mode, in turn fine-tuning differentiated propensity to binary and ternary complex formation, and ultimately cellular degradation activity.

Keywords:  Binding affinity; Conformation; Linker; PROTAC; Stereocenter; VHL

DOI:  https://doi.org/10.1016/j.bmcl.2024.129861
bioRxiv. 2024 Jun 10. pii: 2024.06.10.598126. [Epub ahead of print]

Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.

Kelsey R Baron, Samantha Oviedo, Sophia Krasny, Mashiat Zaman, Rama Aldakhlallah, Prakhyat Mathur, Gerald Pfeffer, Michael J Bollong, Timothy Shutt, Danielle A Grotjahn, R Luke Wiseman.

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic, stress-independent activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic, stress-independent activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that stress-independent activation of these ISR kinases reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic, stress-independent activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

DOI: https://doi.org/10.1101/2024.06.10.598126
PNAS Nexus. 2024 Jun;3(6): pgae229

Gradual ER calcium depletion induces a progressive and reversible UPR signaling.

Ilaria Pontisso, Roberto Ornelas-Guevara, Eric Chevet, Laurent Combettes, Geneviève Dupont.

  The unfolded protein response (UPR) is a widespread signal transduction pathway triggered by endoplasmic reticulum (ER) stress. Because calcium (Ca2+) is a key factor in the maintenance of ER homeostasis, massive Ca2+ depletion of the ER is a potent inducer of ER stress. Although moderate changes in ER Ca2+ drive the ubiquitous Ca2+ signaling pathways, a possible incremental relationship between UPR activation and Ca2+ changes has yet to be described. Here, we determine the sensitivity and time-dependency of activation of the three ER stress sensors, inositol-requiring protein 1 alpha (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 alpha (ATF6α) in response to controlled changes in the concentration of ER Ca2+ in human cultured cells. Combining Ca2+ imaging, fluorescence recovery after photobleaching experiments, biochemical analyses, and mathematical modeling, we uncover a nonlinear rate of activation of the IRE1α branch of UPR, as compared to the PERK and ATF6α branches that become activated gradually with time and are sensitive to more important ER Ca2+ depletions. However, the three arms are all activated within a 1 h timescale. The model predicted the deactivation of PERK and IRE1α upon refilling the ER with Ca2+. Accordingly, we showed that ER Ca2+ replenishment leads to the complete reversion of IRE1α and PERK phosphorylation in less than 15 min, thus revealing the highly plastic character of the activation of the upstream UPR sensors. In conclusion, our results reveal a dynamic and dose-sensitive Ca2+-dependent activation/deactivation cycle of UPR induction, which could tightly control cell fate upon acute and/or chronic stress.

Keywords:  ER stress; UPR reversion; calcium signaling; computational modeling

DOI:  https://doi.org/10.1093/pnasnexus/pgae229
JCI Insight. 2024 Jun 27. pii: e178725. [Epub ahead of print]

A metabolic redox relay supports ER proinsulin export in pancreatic islet β-cells.

Kristen E Rohli, Nicole J Stubbe, Emily M Walker, Gemma L Pearson, Scott A Soleimanpour, Samuel B Stephens.

  Endoplasmic reticulum (ER) stress and proinsulin misfolding are heralded as contributing factors to β-cell dysfunction in Type 2 diabetes (T2D), yet how ER function becomes compromised is not well understood. Recent data identifies altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple β-cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that β-cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas Txnip suppression restored ER redox and proinsulin trafficking. Taken together, we propose that β-cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.

Keywords:  Beta cells; Cell biology; Endocrinology; Insulin; Protein traffic

DOI:  https://doi.org/10.1172/jci.insight.178725
Antioxid Redox Signal. 2024 Jun 25.

Preferential secretion of oxidation-sensitive proteins by unconventional pathways: Why is this important for inflammation?

Marco E Bianchi, Anna Rubartelli, Roberto Sitia.

Significance Fidelity of intercellular communication depends on unambiguous interactions between protein ligands and membrane receptors. Most proteins destined to the extracellular space adopt the required three-dimensional shape as they travel through the endoplasmic reticulum (ER) and Golgi complex and other organelles of the exocytic pathway. However, some proteins, many of which involved in inflammation, avoid this classical secretory route and follow unconventional pathways to leave the cell. Recent advance Stringent quality control systems operate in the ER and cis-Golgi, restricting transport to native conformers, devoid of non-native disulfides and/or reactive thiols. However, some proteins released by living cells require reduced cysteines to exert their extracellular function(s). Remarkably, these proteins lack the secretory signal sequence normally required by secretory proteins for translocation into the ER lumen. Critical issues Why do IL-1β, HMGB1 and other proinflammatory proteins avoid the ER-Golgi route to reach the intercellular space? These proteins require reactive cysteines for exerting their function. Therefore, eluding thiol-mediated quality control along the exocytic pathway is likely one of the main reasons why extracellular proteins that need to be reduced utilize unconventional pathways of secretion, where a quality control aimed at oxidating native cysteines is not present. Future directions Particularly under stress conditions, cells release redox-active enzymes and non-protein thiol compounds that exert an extracellular control of redox-sensitive protein activity, shaping inflammatory responses. This post-secretion, redox-dependent editing of protein messages is still largely undefined. Understanding the underlying mechanistic events will hopefully provide new tools to control inflammation.

DOI: https://doi.org/10.1089/ars.2024.0554
Aging Cell. 2024 Jun 26. e14245

Pathogenic tau induces an adaptive elevation in mRNA translation rate at early stages of disease.

Gabrielle Zuniga, Sakie Katsumura, Jasmine De Mange, Paulino Ramirez, Farzaneh Atrian, Masahiro Morita, Bess Frost.

  Alterations in the rate and accuracy of messenger RNA (mRNA) translation are associated with aging and several neurodegenerative disorders, including Alzheimer's disease and related tauopathies. We previously reported that error-containing RNA that are normally cleared via nonsense-mediated mRNA decay (NMD), a key RNA surveillance mechanism, are translated in the adult brain of a Drosophila model of tauopathy. In the current study, we find that newly-synthesized peptides and translation machinery accumulate within nuclear envelope invaginations that occur as a consequence of tau pathology, and that the rate of mRNA translation is globally elevated in early stages of disease in adult brains of Drosophila models of tauopathy. Polysome profiling from adult heads of tau transgenic Drosophila reveals the preferential translation of specific mRNA that have been previously linked to neurodegeneration. Unexpectedly, we find that panneuronal elevation of NMD further elevates the global translation rate in tau transgenic Drosophila, as does treatment with rapamycin. As NMD activation and rapamycin both suppress tau-induced neurodegeneration, their shared effect on translation suggests that elevated rates of mRNA translation are an early adaptive mechanism to limit neurodegeneration. Our work provides compelling evidence that tau-induced deficits in NMD reshape the tau translatome by increasing translation of RNA that are normally repressed in healthy cells.

Keywords:   Drosophila ; Alzheimer's disease; neurodegeneration; nonsense‐mediated mRNA decay; nucleus; protein translation; tauopathy

DOI:  https://doi.org/10.1111/acel.14245
JCI Insight. 2024 Jun 24. pii: e177743. [Epub ahead of print]9(12):

Protein disulfide isomerase is essential for spermatogenesis in mice.

Yaqiong Zhang, Aizhen Yang, Zhenzhen Zhao, Fengwu Chen, Xiaofeng Yan, Yue Han, Depei Wu, Yi Wu.

  Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.

Keywords:  Cell biology; Protein misfolding; Reproductive biochemistry; Reproductive biology

DOI:  https://doi.org/10.1172/jci.insight.177743
Mol Cancer Ther. 2024 Jun 22.

Discovery of Monovalent Direct Degraders of BRD4 That Act Via the Recruitment of DCAF11.

Gregory S Parker, Julia I Toth, Sarah Fish, Gabrielle A Blanco, Taylor Kampert, Xiaoming Li, Linette Yang, Craig R Stumpf, Kenneth Steadman, Aleksandar Jamborcic, Stephen Chien, Elizabeth Daniele, Alejandro Dearie, Geoffray Leriche, Simon Bailey, Peggy A Thompson.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extra-terminal (BET) protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent anti-tumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent anti-tumor activity in vivo.

DOI: https://doi.org/10.1158/1535-7163.MCT-24-0219
EMBO Rep. 2024 Jun 27.

Recruitment of Ahsa1 to Hsp90 is regulated by a conserved peptide that inhibits ATPase stimulation.

Solomon K Hussein, Rakesh Bhat, Michael Overduin, Paul LaPointe.

  Hsp90 is a molecular chaperone that acts on its clients through an ATP-dependent and conformationally dynamic functional cycle. The cochaperone Accelerator of Hsp90 ATPase, or Ahsa1, is the most potent stimulator of Hsp90 ATPase activity. Ahsa1 stimulates the rate of Hsp90 ATPase activity through a conserved motif, NxNNWHW. Metazoan Ahsa1, but not yeast, possesses an additional 20 amino acid peptide preceding the NxNNWHW motif that we have called the intrinsic chaperone domain (ICD). The ICD of Ahsa1 diminishes Hsp90 ATPase stimulation by interfering with the function of the NxNNWHW motif. Furthermore, the NxNNWHW modulates Hsp90's apparent affinity to Ahsa1 and ATP. Lastly, the ICD controls the regulated recruitment of Hsp90 in cells and its deletion results in the loss of interaction with Hsp90 and the glucocorticoid receptor. This work provides clues to how Ahsa1 conserved regions modulate Hsp90 kinetics and how they may be coupled to client folding status.

Keywords:  ATPase; Ahsa1/Aha1; Chaperones; Hsp90; Protein Folding

DOI:  https://doi.org/10.1038/s44319-024-00193-8
Leukemia. 2024 Jun 22.

Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy.

Céline Philippe, Manon Jaud, Kelly Féral, Alexandre Gay, Loïc Van Den Berghe, Manon Farce, Marina Bousquet, Stéphane Pyronnet, Laurent Mazzolini, Kevin Rouault-Pierre, Christian Touriol.

Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell's viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.

DOI: https://doi.org/10.1038/s41375-024-02321-8
Nat Cell Biol. 2024 Jun 26.

A dual role of ERGIC-localized Rabs in TMED10-mediated unconventional protein secretion.

Yuxin Sun, Xuan Tao, Yaping Han, Xubo Lin, Rui Tian, Haodong Wang, Pei Chang, Qiming Sun, Liang Ge, Min Zhang.

Cargo translocation across membranes is a crucial aspect of secretion. In conventional secretion signal peptide-equipped proteins enter the endoplasmic reticulum (ER), whereas a subset of cargo lacking signal peptides translocate into the ER-Golgi intermediate compartment (ERGIC) in a process called unconventional protein secretion (UcPS). The regulatory events at the ERGIC in UcPS are unclear. Here we reveal the involvement of ERGIC-localized small GTPases, Rab1 (Rab1A and Rab1B) and Rab2A, in regulating UcPS cargo transport via TMED10 on the ERGIC. Rab1 enhances TMED10 translocator activity, promoting cargo translocation into the ERGIC, whereas Rab2A, in collaboration with KIF5B, regulates ERGIC compartmentalization, establishing a UcPS-specific compartment. This study highlights the pivotal role of ERGIC-localized Rabs in governing cargo translocation and specifying the ERGIC's function in UcPS.

DOI: https://doi.org/10.1038/s41556-024-01445-4
Cancer Res. 2024 Jun 26.

Targeting the TRIM14/USP14 Axis Enhances Immunotherapy Efficacy by Inducing Autophagic Degradation of PD-L1.

Di Liu, Mengqiu Li, Zhiyao Zhao, Liang Zhou, Feng Zhi, Zhiyong Guo, Jun Cui.

Immunotherapy has greatly improved cancer treatment in recent years by harnessing the immune system to target cancer cells. The first immunotherapeutic agent approved by the US Food and Drug Administration (FDA) was interferon a (IFNa). Treatment with IFNa can lead to effective immune activation and attenuate tumor immune evasion, but persistent treatment has been shown to elicit immune suppressive effects. Here, we identified an autophagy-dependent mechanism by which IFNa triggers tumor immune evasion by upregulating PD-L1 to suppress the anti-tumor activity of CD8+ T cells. Mechanistically, IFNa increased transcription of TRIM14, which recruited the deubiquitinase USP14 to inhibit the autophagic degradation of PD-L1. USP14 removed K63-linked ubiquitin chains from PD-L1, impairing its recognition by the cargo receptor p62 (also known as SQSTM1) for subsequent autophagic degradation. Combining the USP14 inhibitor IU1 with IFNa and anti-CTLA4 treatment effectively suppressed tumor growth without significant toxicity. This work suggests a strategy for targeting selective autophagy to abolish PD-L1-mediated cancer immune evasion.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-3971
Cell Rep. 2024 Jun 27. pii: S2211-1247(24)00750-2. [Epub ahead of print]43(7): 114421

Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.

Wan-Ping Huang, Brittany C S Ellis, Rachel E Hodgson, Anna Sanchez Avila, Vedanth Kumar, Jessica Rayment, Tobias Moll, Tatyana A Shelkovnikova.

  TDP-43 protein is dysregulated in several neurodegenerative diseases, which often have a multifactorial nature and may have extrinsic stressors as a "second hit." TDP-43 undergoes reversible nuclear condensation in stressed cells including neurons. Here, we demonstrate that stress-inducible nuclear TDP-43 condensates are RNA-depleted, non-liquid assemblies distinct from the known nuclear bodies. Their formation requires TDP-43 oligomerization and ATP and is inhibited by RNA. Using a confocal nanoscanning assay, we find that amyotrophic lateral sclerosis (ALS)-linked mutations alter stress-induced TDP-43 condensation by changing its affinity to liquid-like ribonucleoprotein assemblies. Stress-induced nuclear condensation transiently inactivates TDP-43, leading to loss of interaction with its protein binding partners and loss of function in splicing. Splicing changes are especially prominent and persisting for STMN2 RNA, and STMN2 protein becomes rapidly depleted early during stress. Our results point to early pathological changes to TDP-43 in the nucleus and support therapeutic modulation of stress response in ALS.

Keywords:  ALS; CP: Molecular biology; STMN2; TDP-43; condensate; nuclear body; splicing; stathmin-2; stress

DOI:  https://doi.org/10.1016/j.celrep.2024.114421
Nat Commun. 2024 Jun 25. 15(1): 5359

SDS22 coordinates the assembly of holoenzymes from nascent protein phosphatase-1.

Xinyu Cao, Madryn Lake, Gerd Van der Hoeven, Zander Claes, Javier Del Pino García, Sarah Lemaire, Elora C Greiner, Spyridoula Karamanou, Aleyde Van Eynde, Arminja N Kettenbach, Daniel Natera de Benito, Laura Carrera García, Cristina Hernando Davalillo, Carlos Ortez, Andrés Nascimento, Roser Urreizti, Mathieu Bollen.

SDS22 forms an inactive complex with nascent protein phosphatase PP1 and Inhibitor-3. SDS22:PP1:Inhibitor-3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 stabilizes nascent PP1. In the absence of SDS22, PP1 is gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. Similarly, we identify a female individual with a severe neurodevelopmental disorder bearing an unstable SDS22 mutant, associated with decreased PP1 levels. We furthermore find that SDS22 directly binds to Inhibitor-3 and that this is essential for the stable assembly of SDS22:PP1: Inhibitor-3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled Inhibitor-3 binding site co-transfers with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, through simultaneous interaction with PP1 and Inhibitor-3, integrates the major steps of PP1 holoenzyme assembly.

DOI: https://doi.org/10.1038/s41467-024-49746-4
Cell. 2024 Jun 24. pii: S0092-8674(24)00639-1. [Epub ahead of print]

Microglial-derived C1q integrates into neuronal ribonucleoprotein complexes and impacts protein homeostasis in the aging brain.

Nicole Scott-Hewitt, Matthew Mahoney, Youtong Huang, Nils Korte, T Yvanka de Soysa, Daniel K Wilton, Emily Knorr, Kevin Mastro, Allison Chang, Allison Zhang, David Melville, Monica Schenone, Christina Hartigan, Beth Stevens.

  Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.

Keywords:  C1q; RNA granule; RNA-binding protein; complement; liquid-liquid phase separation; microglia; neuroimmune; neuronal translation; polysome; ribonucleoprotein complex

DOI:  https://doi.org/10.1016/j.cell.2024.05.058
J Cell Sci. 2024 Jun 15. pii: jcs261825. [Epub ahead of print]137(12):

Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention.

Marta Leśniczak-Staszak, Paulina Pietras, Marcin Ruciński, Ryan Johnston, Mateusz Sowiński, Małgorzata Andrzejewska, Michał Nowicki, Ewelina Gowin, Shawn M Lyons, Pavel Ivanov, Witold Szaflarski.

  Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.

Keywords:  Anticancer drugs; EGR1; Lomustine; Stress granules; Translational control; mRNA translation

DOI:  https://doi.org/10.1242/jcs.261825
Cell. 2024 Jun 24. pii: S0092-8674(24)00638-X. [Epub ahead of print]

The extracellular matrix integrates mitochondrial homeostasis.

Hanlin Zhang, C Kimberly Tsui, Gilberto Garcia, Larry K Joe, Haolun Wu, Ayane Maruichi, Wudi Fan, Sentibel Pandovski, Peter H Yoon, Brant M Webster, Jenni Durieux, Phillip A Frankino, Ryo Higuchi-Sanabria, Andrew Dillin.

  Cellular homeostasis is intricately influenced by stimuli from the microenvironment, including signaling molecules, metabolites, and pathogens. Functioning as a signaling hub within the cell, mitochondria integrate information from various intracellular compartments to regulate cellular signaling and metabolism. Multiple studies have shown that mitochondria may respond to various extracellular signaling events. However, it is less clear how changes in the extracellular matrix (ECM) can impact mitochondrial homeostasis to regulate animal physiology. We find that ECM remodeling alters mitochondrial homeostasis in an evolutionarily conserved manner. Mechanistically, ECM remodeling triggers a TGF-β response to induce mitochondrial fission and the unfolded protein response of the mitochondria (UPRMT). At the organismal level, ECM remodeling promotes defense of animals against pathogens through enhanced mitochondrial stress responses. We postulate that this ECM-mitochondria crosstalk represents an ancient immune pathway, which detects infection- or mechanical-stress-induced ECM damage, thereby initiating adaptive mitochondria-based immune and metabolic responses.

Keywords:  TGF-β; TMEM2; extracellular matrix; hyaluronan; immunity; mitochondria

DOI:  https://doi.org/10.1016/j.cell.2024.05.057
Nat Commun. 2024 Jun 25. 15(1): 5388

Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome.

Boyang Zhao, Zhongzheng Cao, Yi Zheng, Phuong Nguyen, Alisa Bowen, Robert H Edwards, Robert M Stroud, Yi Zhou, Menno Van Lookeren Campagne, Fei Li.

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.

DOI: https://doi.org/10.1038/s41467-024-49614-1
Nat Commun. 2024 Jun 25. 15(1): 5360

N-terminal cysteine acetylation and oxidation patterns may define protein stability.

Karen C Heathcote, Thomas P Keeley, Matti Myllykoski, Malin Lundekvam, Nina McTiernan, Salma Akter, Norma Masson, Peter J Ratcliffe, Thomas Arnesen, Emily Flashman.

Oxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.

DOI: https://doi.org/10.1038/s41467-024-49489-2
Nat Cancer. 2024 Jun 28.

The proteogenomic landscape of multiple myeloma reveals insights into disease biology and therapeutic opportunities.

Evelyn Ramberger, Valeriia Sapozhnikova, Yuen Lam Dora Ng, Anna Dolnik, Matthias Ziehm, Oliver Popp, Eric Sträng, Miriam Kull, Florian Grünschläger, Josefine Krüger, Manuela Benary, Sina Müller, Xiang Gao, Arunima Murgai, Mohamed Haji, Annika Schmidt, Raphael Lutz, Axel Nogai, Jan Braune, Dominik Laue, Christian Langer, Cyrus Khandanpour, Florian Bassermann, Hartmut Döhner, Monika Engelhardt, Christian Straka, Michael Hundemer, Dieter Beule, Simon Haas, Ulrich Keller, Hermann Einsele, Lars Bullinger, Stefan Knop, Philipp Mertins, Jan Krönke.

Multiple myeloma (MM) is a plasma cell malignancy of the bone marrow. Despite therapeutic advances, MM remains incurable, and better risk stratification as well as new therapies are therefore highly needed. The proteome of MM has not been systematically assessed before and holds the potential to uncover insight into disease biology and improved prognostication in addition to genetic and transcriptomic studies. Here we provide a comprehensive multiomics analysis including deep tandem mass tag-based quantitative global (phospho)proteomics, RNA sequencing, and nanopore DNA sequencing of 138 primary patient-derived plasma cell malignancies encompassing treatment-naive MM, plasma cell leukemia and the premalignancy monoclonal gammopathy of undetermined significance, as well as healthy controls. We found that the (phospho)proteome of malignant plasma cells are highly deregulated as compared with healthy plasma cells and is both defined by chromosomal alterations as well as posttranscriptional regulation. A prognostic protein signature was identified that is associated with aggressive disease independent of established risk factors in MM. Integration with functional genetics and single-cell RNA sequencing revealed general and genetic subtype-specific deregulated proteins and pathways in plasma cell malignancies that include potential targets for (immuno)therapies. Our study demonstrates the potential of proteogenomics in cancer and provides an easily accessible resource for investigating protein regulation and new therapeutic approaches in MM.

DOI: https://doi.org/10.1038/s43018-024-00784-3
Nat Commun. 2024 Jun 26. 15(1): 5410

METTL3-mediated chromatin contacts promote stress granule phase separation through metabolic reprogramming during senescence.

Chen Wang, Hideki Tanizawa, Connor Hill, Aaron Havas, Qiang Zhang, Liping Liao, Xue Hao, Xue Lei, Lu Wang, Hao Nie, Yuan Qi, Bin Tian, Alessandro Gardini, Andrew V Kossenkov, Aaron Goldman, Shelley L Berger, Ken-Ichi Noma, Peter D Adams, Rugang Zhang.

METTL3 is the catalytic subunit of the methyltransferase complex, which mediates m6A modification to regulate gene expression. In addition, METTL3 regulates transcription in an enzymatic activity-independent manner by driving changes in high-order chromatin structure. However, how these functions of the methyltransferase complex are coordinated remains unknown. Here we show that the methyltransferase complex coordinates its enzymatic activity-dependent and independent functions to regulate cellular senescence, a state of stable cell growth arrest. Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression through the three-dimensional chromatin organization during senescence. Elevated Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, manifesting as stress granule phase separation, by driving metabolic reprogramming. This correlates with an impairment of translation of cell-cycle related mRNAs harboring polymethylated m6A sites. In summary, our results report a coordination of m6A-dependent and -independent function of the methyltransferase complex in regulating senescence through phase separation driven by metabolic reprogramming.

DOI: https://doi.org/10.1038/s41467-024-49745-5
Cell. 2024 Jun 17. pii: S0092-8674(24)00583-X. [Epub ahead of print]

Pan-cancer proteogenomics expands the landscape of therapeutic targets.

Sara R Savage, Xinpei Yi, Jonathan T Lei, Bo Wen, Hongwei Zhao, Yuxing Liao, Eric J Jaehnig, Lauren K Somes, Paul W Shafer, Tobie D Lee, Zile Fu, Yongchao Dou, Zhiao Shi, Daming Gao, Valentina Hoyos, Qiang Gao, Bing Zhang.

  Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development.

Keywords:  pan-cancer, proteogenomics, drug targets, proteomics, synthetic lethality, neoantigens, tumor antigens, data integration

DOI:  https://doi.org/10.1016/j.cell.2024.05.039