bims-proteo 2023-06-25 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–06–25
34 papers selected by
Eric Chevet, INSERM

Mammalian cargo receptors for endoplasmic reticulum-to-Golgi transport: mechanisms and interactions.
Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.
Lipid biosynthesis perturbation impairs Endoplasmic Reticulum-Associated Degradation.
The ER folding sensor UGGT1 acts on TAPBPR-chaperoned peptide-free MHC I.
Perturbation of endoplasmic reticulum proteostasis triggers tissue injury in the thyroid gland.
Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.
PARK15/FBXO7 is dispensable for PINK1/Parkin mitophagy in iNeurons and HeLa cell systems.
SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex.
NAC controls cotranslational N-terminal methionine excision in eukaryotes.
The E3 Ubiquitin Ligase, CHIP/STUB1, Inhibits Aggregation of Phosphorylated Proteoforms of Microtubule-associated Protein Tau (MAPT).
Art2 mediates selective endocytosis of methionine transporters during adaptation to sphingolipid depletion.
Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex.
Nuanced role for dendritic cell intrinsic IRE1 RNase in the regulation of antitumor adaptive immunity.
Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.
Measurement of SQSTM1 by flow cytometry.
The E3 Ligase Poe Promotes Pericentrin Degradation.
Generation of host-directed and virus-specific antivirals using targeted protein degradation promoted by small molecules and viral RNA mimics.
Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.
The ER calcium channel Csg2 integrates sphingolipid metabolism with autophagy.
Control of SOX2 protein stability and tumorigenic activity by E3 ligase CHIP in esophageal cancer cells.
Profiling dynamic RNA-protein interactions using small-molecule-induced RNA editing.
A Golgi-resident GPR108 cooperates with E3 ubiquitin ligase Smurf1 to suppress antiviral innate immunity.
NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells and restrains PDAC initiation.
Trans-Golgi protein TVP23B regulates host-microbe interactions via Paneth cell homeostasis and Goblet cell glycosylation.
Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.
UBE2M-mediated neddylation of TRIM21 regulates obesity-induced inflammation and metabolic disorders.
Quantitative Proteomics-Based Substrate Screening Revealed Cyclophilin Stabilization Regulated by Deubiquitinase Ubp7.
Proteostatic tuning underpins the evolution of novel multicellular traits.
J-domain proteins form binary complexes with Hsp90 and ternary complexes with Hsp90 and Hsp70.
SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.
Proteomic Dynamics of Breast Cancer Cell Lines Identifies Potential Therapeutic Protein Targets.
How soluble misfolded proteins bypass chaperones at the molecular level.
Modulation of cellular metabolism by protein crotonylation regulates pancreatic cancer progression.
Mechanistic insights into the aggregation pathway of the patient-derived immunoglobulin light chain variable domain protein FOR005.

Biochem Soc Trans. 2023 Jun 19. pii: BST20220713. [Epub ahead of print]

Mammalian cargo receptors for endoplasmic reticulum-to-Golgi transport: mechanisms and interactions.

Yuan Zhang, Vishal Srivastava, Bin Zhang.

  Proteins that are destined to enter the secretory pathway are synthesized on the rough endoplasmic reticulum (ER) and then translocated into the ER lumen, where they undergo posttranslational modifications, folding, and assembly. After passing a quality control system, the cargo proteins are packaged into coat protein complex II (COPII) vesicles to exit the ER. In metazoans, most COPII subunits have multiple paralogs, enabling COPII vesicles the flexibility to transport a diverse range of cargo. The cytoplasmic domains of transmembrane proteins can interact with SEC24 subunits of COPII to enter the ER exit sites. Some transmembrane proteins may also act as cargo receptors that bind soluble secretory proteins within the ER lumen, enabling them to enter COPII vesicles. The cytoplasmic domains of cargo receptors also contain coat protein complex I binding motifs that allow for their cycling back to the ER after unloading their cargo in the ER-Golgi intermediate compartment and cis-Golgi. Once unloaded, the soluble cargo proteins continue maturation through the Golgi before reaching their final destinations. This review provides an overview of receptor-mediated transport of secretory proteins from the ER to the Golgi, with a focus on the current understanding of two mammalian cargo receptors: the LMAN1-MCFD2 complex and SURF4, and their roles in human health and disease.

Keywords:  LMAN1–MCFD2; SURF4; endoplasmic reticulum; glycoproteins; golgi apparatus; trafficking

DOI:  https://doi.org/10.1042/BST20220713
Cell. 2023 Jun 13. pii: S0092-8674(23)00587-1. [Epub ahead of print]

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Martin B D Müller, Prasad Kasturi, Gopal G Jayaraj, F Ulrich Hartl.

  Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

Keywords:  BAG6 complex; CCR4/NOT; GCN1; aging; codon optimality; collagens; cotranslational mRNA decay; disomes; readthrough mitigation; transmembrane proteins

DOI:  https://doi.org/10.1016/j.cell.2023.05.035
J Biol Chem. 2023 Jun 16. pii: S0021-9258(23)01967-1. [Epub ahead of print] 104939

Lipid biosynthesis perturbation impairs Endoplasmic Reticulum-Associated Degradation.

Samantha M Turk, Christopher J Indovina, Jacob M Miller, Danielle L Overton, Avery M Runnebohm, Cade J Orchard, Mary E Tragesser-Tiña, Samantha K Gosser, Ellen M Doss, Kyle A Richards, Courtney Broshar Irelan, Mahmoud M Daraghmi, Connor G Bailey, Julia M Niekamp, Kieran P Claypool, Sarah M Engle, Bryce W Buchanan, Kelsey A Woodruff, James B Olesen, Philip J Smaldino, Eric M Rubenstein.

  The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.

Keywords:  Doa10; ER quality control; Hrd1; Saccharomyces cerevisiae; endoplasmic reticulum-associated degradation (ERAD); phospholipid metabolism; protein degradation; sterol; translocon quality control; yeast genetics

DOI:  https://doi.org/10.1016/j.jbc.2023.104939
Elife. 2023 Jun 22. pii: e85432. [Epub ahead of print]12

The ER folding sensor UGGT1 acts on TAPBPR-chaperoned peptide-free MHC I.

Lina Sagert, Christian Winter, Ina Ruppert, Maximilian Zehetmaier, Christoph Thomas, Robert Tampé.

  Adaptive immune responses are triggered by antigenic peptides presented on major histocompatibility complex class I (MHC I) at the surface of pathogen-infected or cancerous cells. Formation of stable peptide-MHC I complexes is facilitated by tapasin and TAPBPR, two related MHC I-specific chaperones that catalyze selective loading of suitable peptides onto MHC I in a process called peptide editing or proofreading. On their journey to the cell surface, MHC I complexes must pass a quality control step performed by UGGT1, which senses the folding status of the transiting N-linked glycoproteins in the endoplasmic reticulum (ER). UGGT1 reglucosylates non-native glycoproteins and thereby allows them to revisit the ER folding machinery. Here, we describe a reconstituted in-vitro system of purified human proteins that enabled us to delineate the function of TAPBPR during the UGGT1-catalyzed quality control and reglucosylation of MHC I. By combining glycoengineering with liquid chromatography-mass spectrometry, we show that TAPBPR promotes reglucosylation of peptide-free MHC I by UGGT1. Thus, UGGT1 cooperates with TAPBPR in fulfilling a crucial function in the quality control mechanisms of antigen processing and presentation.

Keywords:  biochemistry; chemical biology; human; immunology; inflammation

DOI:  https://doi.org/10.7554/eLife.85432
JCI Insight. 2023 06 22. pii: e169937. [Epub ahead of print]8(12):

Perturbation of endoplasmic reticulum proteostasis triggers tissue injury in the thyroid gland.

Xiaohan Zhang, Crystal Young, Xiao-Hui Liao, Samuel Refetoff, Mauricio Torres, Yaron Tomer, Mihaela Stefan-Lifshitz, Hao Zhang, Dennis Larkin, Deyu Fang, Ling Qi, Peter Arvan.

  Defects in endoplasmic reticulum (ER) proteostasis have been linked to diseases in multiple organ systems. Here we examined the impact of perturbation of ER proteostasis in mice bearing thyrocyte-specific knockout of either HRD1 (to disable ER-associated protein degradation [ERAD]) or ATG7 (to disable autophagy) in the absence or presence of heterozygous expression of misfolded mutant thyroglobulin (the most highly expressed thyroid gene product, synthesized in the ER). Misfolding-inducing thyroglobulin mutations are common in humans but are said to yield only autosomal-recessive disease - perhaps because misfolded thyroglobulin protein might undergo disposal by ERAD or ER macroautophagy. We find that as single defects, neither ERAD, nor autophagy, nor heterozygous thyroglobulin misfolding altered circulating thyroxine levels, and neither defective ERAD nor defective autophagy caused any gross morphological change in an otherwise WT thyroid gland. However, heterozygous expression of misfolded thyroglobulin itself triggered significant ER stress and individual thyrocyte death while maintaining integrity of the surrounding thyroid epithelium. In this context, deficiency of ERAD (but not autophagy) resulted in patchy whole-follicle death with follicular collapse and degeneration, accompanied by infiltration of bone marrow-derived macrophages. Perturbation of thyrocyte ER proteostasis is thus a risk factor for both cell death and follicular demise.

Keywords:  Cell Biology; Protein misfolding

DOI:  https://doi.org/10.1172/jci.insight.169937
bioRxiv. 2023 Jun 07. pii: 2023.06.07.544135. [Epub ahead of print]

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

Yi-Ju Tseng, Indranil Malik, Xiexiong Deng, Amy Krans, Karen Jansen-West, Elizabeth M H Tank, Nicolas B Gomez, Roger Sher, Leonard Petrucelli, Sami J Barmada, Peter K Todd.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1, and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter cell lines and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation - suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation elongation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

DOI: https://doi.org/10.1101/2023.06.07.544135
EMBO Rep. 2023 Jun 19. e56399

PARK15/FBXO7 is dispensable for PINK1/Parkin mitophagy in iNeurons and HeLa cell systems.

Felix Kraus, Ellen A Goodall, Ian R Smith, Yizhi Jiang, Julia C Paoli, Frank Adolf, Jiuchun Zhang, Joao A Paulo, Brenda A Schulman, J Wade Harper.

  The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation (pUb), Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support the recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization and mt UPR-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/- cells have no demonstrable defect in: (i) kinetics of pUb accumulation, (ii) pUb puncta on mitochondria by super-resolution imaging, (iii) recruitment of Parkin and autophagy machinery to damaged mitochondria, (iv) mitophagic flux, and (v) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.

Keywords:  FBXO7; iNeurons; mitophagy; proteomics; ubiquitin ligase

DOI:  https://doi.org/10.15252/embr.202256399
bioRxiv. 2023 Jun 07. pii: 2023.04.13.536796. [Epub ahead of print]

SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex.

Liangguang Leo Lin, Xiaoqiong Wei, Huilun Helen Wang, Brent Pederson, Mauricio Torres, You Lu, Zexin Jason Li, Xiaodan Liu, Hancheng Mao, Hui Wang, Zhen Zhao, Shengyi Sun, Ling Qi.

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD); however, definitive evidence for the importance of SEL1L in HRD1 ERAD is lacking. Here we report that attenuation of the interaction between SEL1L and HRD1 impairs HRD1 ERAD function and has pathological consequences in mice. Our data show that SEL1L variant p.Ser658Pro ( SEL1L S 658 P ) previously identified in Finnish Hound suffering cerebellar ataxia is a recessive hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Mechanistically, SEL1L S 658 P variant attenuates the SEL1L-HRD1 interaction and causes HRD1 dysfunction by generating electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes revealed that the SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L recruits not only the lectins OS9 and ERLEC1, but the E2 UBE2J1 and retrotranslocon DERLIN, to HRD1. These data underscore the pathophysiological importance and disease relevance of the SEL1L-HRD1 complex, and identify a key step in organizing the HRD1 ERAD complex.

DOI: https://doi.org/10.1101/2023.04.13.536796
Science. 2023 Jun 23. 380(6651): 1238-1243

NAC controls cotranslational N-terminal methionine excision in eukaryotes.

Martin Gamerdinger, Min Jia, Renate Schloemer, Laurenz Rabl, Mateusz Jaskolowski, Katrin M Khakzar, Zeynel Ulusoy, Annalena Wallisch, Ahmad Jomaa, Gundula Hunaeus, Alain Scaiola, Kay Diederichs, Nenad Ban, Elke Deuerling.

N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1. NAC recruits METAP1 using a long, flexible tail and provides a platform for the formation of an active methionine excision complex at the ribosomal tunnel exit. This mode of interaction ensures the efficient excision of methionine from cytosolic proteins, whereas proteins targeted to the endoplasmic reticulum are spared. Our results suggest a broader mechanism for how access of protein biogenesis factors to translating ribosomes is controlled.

DOI: https://doi.org/10.1126/science.adg3297
J Mol Biol. 2023 Mar 09. pii: S0022-2836(23)00082-7. [Epub ahead of print] 168026

The E3 Ubiquitin Ligase, CHIP/STUB1, Inhibits Aggregation of Phosphorylated Proteoforms of Microtubule-associated Protein Tau (MAPT).

Cory M Nadel, Aye C Thwin, Matthew Callahan, Kanghyun Lee, Emily Connelly, Charles S Craik, Daniel R Southworth, Jason E Gestwicki.

  Hyper-phosphorylated tau accumulates as insoluble fibrils in Alzheimer's disease (AD) and related dementias. The strong correlation between phosphorylated tau and disease has led to an interest in understanding how cellular factors discriminate it from normal tau. Here, we screen a panel of chaperones containing tetratricopeptide repeat (TPR) domains to identify those that might selectively interact with phosphorylated tau. We find that the E3 ubiquitin ligase, CHIP/STUB1, binds 10-fold more strongly to phosphorylated tau than unmodified tau. The presence of even sub-stoichiometric concentrations of CHIP strongly suppresses aggregation and seeding of phosphorylated tau. We also find that CHIP promotes rapid ubiquitination of phosphorylated tau, but not unmodified tau, in vitro. Binding to phosphorylated tau requires CHIP's TPR domain, but the binding mode is partially distinct from the canonical one. In cells, CHIP restricts seeding by phosphorylated tau, suggesting that it could be an important barrier in cell-to-cell spreading. Together, these findings show that CHIP recognizes a phosphorylation-dependent degron on tau, establishing a pathway for regulating the solubility and turnover of this pathological proteoform.

Keywords:  intrinsically disordered protein; phospho-degrons; protein aggregation; protein–protein interactions; tauopathy

DOI:  https://doi.org/10.1016/j.jmb.2023.168026
J Cell Sci. 2023 Jun 20. pii: jcs.260675. [Epub ahead of print]

Art2 mediates selective endocytosis of methionine transporters during adaptation to sphingolipid depletion.

Nathaniel L Hepowit, Bradley Moon, Adam C Ebert, Robert C Dickson, Jason A MacGurn.

  Accumulating evidence in several model organisms indicates that reduced sphingolipid biosynthesis promotes longevity, although underlying mechanisms remain unclear. In yeast, sphingolipid depletion induces a state resembling amino acid restriction, which we hypothesized may be due to altered stability of amino acid transporters at the plasma membrane. To test this, we measured surface abundance for a diverse panel of membrane proteins in the presence of myriocin, a sphingolipid biosynthesis inhibitor. Unexpectedly, we found that surface levels of most proteins examined were either unaffected or increased during myriocin treatment, consistent with an observed decrease in bulk endocytosis. In contrast, sphingolipid depletion triggered selective endocytosis of the methionine transporter Mup1. Unlike methionine-induced Mup1 endocytosis, myriocin triggers Mup1 endocytosis that requires the Rsp5 adaptor Art2, C-terminal lysine residues, and the formation of K63-linked ubiquitin polymers. These findings reveal cellular adaptation to sphingolipid depletion by ubiquitin-mediated remodeling of nutrient transporter composition at the cell surface.

Keywords:  Alpha-arrestins; Amino acid transporters; Endocytic adaptors; Endocytosis; Glucose transport; Methionine transport; Myriocin; Sphingolipid metabolism; Ubiquitin

DOI:  https://doi.org/10.1242/jcs.260675
Nat Commun. 2023 Jun 23. 14(1): 3762

Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex.

Yilun Sun, Simone A Baechler, Xiaohu Zhang, Suresh Kumar, Valentina M Factor, Yasuhiro Arakawa, Cindy H Chau, Kanako Okamoto, Anup Parikh, Bob Walker, Yijun P Su, Jiji Chen, Tabitha Ting, Shar-Yin N Huang, Erin Beck, Zina Itkin, Crystal McKnight, Changqing Xie, Nitin Roper, Deepak Nijhawan, William Douglas Figg, Paul S Meltzer, James C Yang, Craig J Thomas, Yves Pommier.

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

DOI: https://doi.org/10.1038/s41467-023-39374-9
Front Immunol. 2023 ;14 1209588

Nuanced role for dendritic cell intrinsic IRE1 RNase in the regulation of antitumor adaptive immunity.

Felipe Flores-Santibañez, Sofie Rennen, Dominique Fernández, Clint De Nolf, Evelien Van De Velde, Sandra Gaete González, Camila Fuentes, Carolina Moreno, Diego Figueroa, Álvaro Lladser, Takao Iwawaki, María Rosa Bono, Sophie Janssens, Fabiola Osorio.

  In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.

Keywords:  IRE1; XBP1; antitumor immune response; cDC1; dendritic cells; immunity; melanoma; unfolded protein response

DOI:  https://doi.org/10.3389/fimmu.2023.1209588
Sci Adv. 2023 Jun 23. 9(25): eade7890

Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.

Jennifer L Mamrosh, David J Sherman, Joseph R Cohen, James A Johnston, Marisa K Joubert, Jing Li, J Russell Lipford, Brett Lomenick, Annie Moradian, Siddharth Prabhu, Michael J Sweredoski, Bryan Vander Lugt, Rati Verma, Raymond J Deshaies.

Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, enriched in peptides of mitochondrial origin, appears independent of the UPS. A further ~10% of peptides were found to be dependent on the proteasome but independent of ubiquitination for their generation. Notably, clinically achievable partial inhibition of the proteasome resulted in display of atypical peptides. Our results suggest that generation of MHC class I•peptide complexes is more complex than previously recognized, with UPS-dependent and UPS-independent components; paradoxically, alternative protein degradation pathways also generate class I peptides when canonical pathways are impaired.

DOI: https://doi.org/10.1126/sciadv.ade7890
Autophagy. 2023 Jun 19. 1-11

Measurement of SQSTM1 by flow cytometry.

Jessica C Hargarten, Guowu Hu, Waleed Elsegeiny, Peter R Williamson.

  Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A1/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux.Abbreviations: ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type.

Keywords:  Autophagy; Bafilomycin A1; Sqstm1/P62; chloroquine; flow cytometry; serum starvation

DOI:  https://doi.org/10.1080/15548627.2023.2224074
Mol Biol Cell. 2023 Jun 21. mbcE22110534

The E3 Ligase Poe Promotes Pericentrin Degradation.

Brian J Galletta, Ramya Varadarajan, Carey J Fagerstrom, Bing Yang, Karen Plevock Haase, Katherine McJunkin, Nasser M Rusan.

Centrosomes are essential parts of diverse cellular processes and precise regulation of the levels of their constituent proteins is critical for their function. One such protein is Pericentrin - PCNT in humans and PLP in Drosophila. Increased PCNT expression and its protein accumulation is linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remain underexplored. Our previous study (Galletta et al., 2020) demonstrated that PLP levels are sharply downregulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. We hypothesized that the sharp drop in PLP protein was a result of rapid protein degradation during the male germline pre-meiotic G2 phase. Here we show that PLP is subject to ubiquitin-mediated degradation and identify multiple proteins that promote the reduction of PLP levels in spermatocytes, including the UBR box containing E3 ligase Poe (UBR4), which we show binds to PLP. While protein sequences governing post-translational regulation of PLP are not restricted to a single region of the protein, we identify a region that is required for Poe-mediated degradation. Experimentally stabilizing PLP, via internal PLP deletions or loss of Poe, leads to PLP accumulation in spermatocytes, its mispositioning along centrioles, and defects in centriole docking in spermatids.

DOI: https://doi.org/10.1091/mbc.E22-11-0534
Cell Host Microbe. 2023 Jun 05. pii: S1931-3128(23)00223-8. [Epub ahead of print]

Generation of host-directed and virus-specific antivirals using targeted protein degradation promoted by small molecules and viral RNA mimics.

Nan Zhao, Jessica Sook Yuin Ho, Fanye Meng, Simin Zheng, Andrew P Kurland, Lu Tian, Martha Rea-Moreno, Xiangyang Song, Ji-Seon Seo, H Ümit Kaniskan, Aartjan J W Te Velthuis, Domenico Tortorella, Ya-Wen Chen, Jeffrey R Johnson, Jian Jin, Ivan Marazzi.

  Targeted protein degradation (TPD), as exemplified by proteolysis-targeting chimera (PROTAC), is an emerging drug discovery platform. PROTAC molecules, which typically contain a target protein ligand linked to an E3 ligase ligand, recruit a target protein to the E3 ligase to induce its ubiquitination and degradation. Here, we applied PROTAC approaches to develop broad-spectrum antivirals targeting key host factors for many viruses and virus-specific antivirals targeting unique viral proteins. For host-directed antivirals, we identified a small-molecule degrader, FM-74-103, that elicits selective degradation of human GSPT1, a translation termination factor. FM-74-103-mediated GSPT1 degradation inhibits both RNA and DNA viruses. Among virus-specific antivirals, we developed viral RNA oligonucleotide-based bifunctional molecules (Destroyers). As a proof of principle, RNA mimics of viral promoter sequences were used as heterobifunctional molecules to recruit and target influenza viral polymerase for degradation. This work highlights the broad utility of TPD to rationally design and develop next-generation antivirals.

Keywords:  CMV; GSPT1; PROTAC; SARS-CoV-2; antiviral therapeutics; influenza virus; oligonucleotide; small molecule

DOI:  https://doi.org/10.1016/j.chom.2023.05.030
J Biol Chem. 2023 Jun 20. pii: S0021-9258(23)01973-7. [Epub ahead of print] 104945

Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.

Justin C Rudd, Sibaprasad Maity, James A Grunkemeyer, Joshua C Snyder, Sándor Lovas, Laura A Hansen.

  Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness-comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant of human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Lastly, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2 and Dynamin-1 dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes.

Keywords:  Flower; cell competition; endocytosis; live-cell imaging; membrane protein structure; molecular dynamics simulations

DOI:  https://doi.org/10.1016/j.jbc.2023.104945
Nat Commun. 2023 Jun 22. 14(1): 3725

The ER calcium channel Csg2 integrates sphingolipid metabolism with autophagy.

Shiyan Liu, Mutian Chen, Yichang Wang, Yuqing Lei, Ting Huang, Yabin Zhang, Sin Man Lam, Huihui Li, Shiqian Qi, Jia Geng, Kefeng Lu.

Sphingolipids are ubiquitous components of membranes and function as bioactive lipid signaling molecules. Here, through genetic screening and lipidomics analyses, we find that the endoplasmic reticulum (ER) calcium channel Csg2 integrates sphingolipid metabolism with autophagy by regulating ER calcium homeostasis in the yeast Saccharomyces cerevisiae. Csg2 functions as a calcium release channel and maintains calcium homeostasis in the ER, which enables normal functioning of the essential sphingolipid synthase Aur1. Under starvation conditions, deletion of Csg2 causes increases in calcium levels in the ER and then disturbs Aur1 stability, leading to accumulation of the bioactive sphingolipid phytosphingosine, which specifically and completely blocks autophagy and induces loss of starvation resistance in cells. Our findings indicate that calcium homeostasis in the ER mediated by the channel Csg2 translates sphingolipid metabolism into autophagy regulation, further supporting the role of the ER as a signaling hub for calcium homeostasis, sphingolipid metabolism and autophagy.

DOI: https://doi.org/10.1038/s41467-023-39482-6
Oncogene. 2023 Jun 23.

Control of SOX2 protein stability and tumorigenic activity by E3 ligase CHIP in esophageal cancer cells.

Li Kang, Huifang Zhang, Yaling Wang, Manyu Chu, Jianzhong He, Mengyang Xue, Liu Pan, Yunfeng Zhang, Zhen Wang, Zhaosu Chen, Yuanyong Huang, Zitai Chen, Enmin Li, Jiwen Li, Liyan Xu, Rong Zhang, Jiemin Wong.

SOX2 is highly expressed and controls tumor initiation and cancer stem cell function in various squamous cell carcinomas including esophageal squamous cancer. However, the molecular mechanism leading to SOX2 overexpression in cancer is incompletely understood. Here, we identified CHIP, a chaperone-associated ubiquitin E3 ligase, as a novel negative regulator of SOX2 protein stability and tumorigenic activity in esophageal squamous carcinoma cells. We showed that CHIP interacted with SOX2 primarily via chaperone HSP70, together they catalyzed SOX2 ubiquitination and degradation via proteasome. In contrast, HSP90 promoted SOX2 stability and inhibition of HSP90 activity induced SOX2 ubiquitination and degradation. Notably, unlike the case in normal esophageal tissues where CHIP was detected in both the cytoplasm and nucleus, CHIP in clinical esophageal tumor specimens was predominantly localized in the cytoplasm. Consistent with this observation, we observed increased expression of exportin-1/CRM-1 in clinical esophageal tumor specimens. We further demonstrated that CHIP catalyzed SOX2 ubiquitination and degradation primarily in the nuclear compartment. Taken together, our study has identified CHIP as a key suppressor of SOX2 protein stability and tumorigenic activity and revealed CHIP nuclear exclusion as a potential mechanism for aberrant SOX2 overexpression in esophageal cancer. Our study also suggests HSP90 inhibitors as potential therapeutic agents for SOX2-positive cancers.

DOI: https://doi.org/10.1038/s41388-023-02745-z
Nat Chem Biol. 2023 Jun 22.

Profiling dynamic RNA-protein interactions using small-molecule-induced RNA editing.

Kyung W Seo, Ralph E Kleiner.

RNA-binding proteins (RBPs) play an important role in biology, and characterizing dynamic RNA-protein interactions is essential for understanding RBP function. In this study, we developed targets of RBPs identified by editing induced through dimerization (TRIBE-ID), a facile strategy for quantifying state-specific RNA-protein interactions upon rapamycin-mediated chemically induced dimerization and RNA editing. We performed TRIBE-ID with G3BP1 and YBX1 to study RNA-protein interactions during normal conditions and upon oxidative stress-induced biomolecular condensate formation. We quantified editing kinetics to infer interaction persistence and show that stress granule formation strengthens pre-existing RNA-protein interactions and induces new RNA-protein binding events. Furthermore, we demonstrate that G3BP1 stabilizes its targets under normal and oxidative stress conditions independent of stress granule formation. Finally, we apply our method to characterize small-molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile dynamic RNA-protein interactions in cellular contexts with temporal control.

DOI: https://doi.org/10.1038/s41589-023-01372-9
Cell Rep. 2023 Jun 17. pii: S2211-1247(23)00666-6. [Epub ahead of print]42(6): 112655

A Golgi-resident GPR108 cooperates with E3 ubiquitin ligase Smurf1 to suppress antiviral innate immunity.

Mengyuan Zhao, Yong Zhang, Lihua Qiang, Zhe Lu, Zhuo Zhao, Yesheng Fu, Bo Wu, Qiyao Chai, Pupu Ge, Zehui Lei, Xinwen Zhang, Bingxi Li, Jing Wang, Lingqiang Zhang, Cui Hua Liu.

  The regulation of antiviral immunity is crucial in maintaining host immune homeostasis, a process that involves dynamic modulations of host organelles. The Golgi apparatus is increasingly perceived as a host organelle functioning as a critical platform for innate immunity, but the detailed mechanism by which it regulates antiviral immunity remains elusive. Here, we identify the Golgi-localized G protein-coupled receptor 108 (GPR108) as a regulator of type Ι interferon responses by targeting interferon regulatory factor 3 (IRF3). Mechanistically, GPR108 enhances the ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1)-mediated K63-linked polyubiquitination of phosphorylated IRF3 for nuclear dot 10 protein 52 (NDP52)-dependent autophagic degradation, leading to suppression of antiviral immune responses against DNA or RNA viruses. Taken together, our study provides insight into the crosstalk between the Golgi apparatus and antiviral immunity via a dynamic and spatiotemporal regulation of GPR108-Smurf1 axis, thereby indicating a potential target for treating viral infection.

Keywords:  CP: Immunology; GPR108; Golgi apparatus; IRF3; Smurf1; antiviral innate immunity; autophagy; ubiquitination

DOI:  https://doi.org/10.1016/j.celrep.2023.112655
Nat Commun. 2023 Jun 23. 14(1): 3761

NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells and restrains PDAC initiation.

Isidoro Cobo, Sumit Paliwal, Cristina Bodas, Irene Felipe, Júlia Melià-Alomà, Ariadna Torres, Jaime Martínez-Villarreal, Marina Malumbres, Fernando García, Irene Millán, Natalia Del Pozo, Joo-Cheol Park, Ray J MacDonald, Javier Muñoz, Raúl Méndez, Francisco X Real.

Pancreatic acinar cells rely on PTF1 and other transcription factors to deploy their transcriptional program. We identify NFIC as a NR5A2 interactor and regulator of acinar differentiation. NFIC binding sites are enriched in NR5A2 ChIP-Sequencing peaks. Nfic knockout mice have a smaller, histologically normal, pancreas with reduced acinar gene expression. NFIC binds and regulates the promoters of acinar genes and those involved in RNA/protein metabolism, and Nfic knockout pancreata show defective ribosomal RNA maturation. NFIC dampens the endoplasmic reticulum stress program through binding to gene promoters and is required for resolution of Tunicamycin-mediated stress. NFIC is down-regulated during caerulein pancreatitis and is required for recovery after damage. Normal human pancreata with low levels of NFIC transcripts display reduced expression of genes down-regulated in Nfic knockout mice. NFIC expression is down-regulated in mouse and human pancreatic ductal adenocarcinoma. Consistently, Nfic knockout mice develop a higher number of mutant Kras-driven pre-neoplastic lesions.

DOI: https://doi.org/10.1038/s41467-023-39291-x
Nat Commun. 2023 Jun 20. 14(1): 3652

Trans-Golgi protein TVP23B regulates host-microbe interactions via Paneth cell homeostasis and Goblet cell glycosylation.

Ran Song, William McAlpine, Aaron M Fond, Evan Nair-Gill, Jin Huk Choi, Elisabeth E L Nyström, Liisa Arike, Sydney Field, Xiaohong Li, Jeffrey A SoRelle, James J Moresco, Eva Marie Y Moresco, John R Yates, Parastoo Azadi, Josephine Ni, George M H Birchenough, Bruce Beutler, Emre E Turer.

A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.

DOI: https://doi.org/10.1038/s41467-023-39398-1
Neurobiol Dis. 2023 Jun 21. pii: S0969-9961(23)00227-9. [Epub ahead of print] 106212

Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.

Indranil Malik, Yi-Ju Tseng, Clare Wieland, Katelyn M Green, Kristina Zheng, Katyanne Calleja, Peter K Todd.

  Neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by a CGG trinucleotide repeat expansion in the 5' UTR of FMR1. Expanded CGG repeat RNAs form stable secondary structures, which in turn support repeat-associated non-AUG (RAN) translation to produce toxic peptides. The parameters that impact RAN translation initiation efficiency are not well understood. Here we used a Drosophila melanogaster model of FXTAS to evaluate the role of the eIF4G family of eukaryotic translation initiation factors (EIF4G1, EIF4GII and EIF4G2/DAP5) in modulating RAN translation and CGG repeat-associated toxicity. DAP5 knockdown robustly suppressed CGG repeat-associated toxicity and inhibited RAN translation. Furthermore, knockdown of initiation factors that preferentially associate with DAP5 (such as EIF2β, EIF3F and EIF3G) also selectively suppressed CGG repeat-induced eye degeneration. In mammalian cellular reporter assays, DAP5 knockdown exhibited modest and cell-type specific effects on RAN translation. Taken together, these data support a role for DAP5 in CGG repeat associated toxicity possibly through modulation of RAN translation.

Keywords:  DAP5; FXTAS; Fragile X; RAN translation; repeat expansion disorders

DOI:  https://doi.org/10.1016/j.nbd.2023.106212
Cell Metab. 2023 Jun 14. pii: S1550-4131(23)00209-7. [Epub ahead of print]

UBE2M-mediated neddylation of TRIM21 regulates obesity-induced inflammation and metabolic disorders.

Xinliang Lu, Xianghui Kong, Hao Wu, Jiayue Hao, Sirui Li, Zichun Gu, Xianchang Zeng, Yingying Shen, Shibo Wang, Jiming Chen, Xuefeng Fei, Yi Sun, Xu Li, Lingling Jiang, Fei Yang, Jianli Wang, Zhijian Cai.

  Inflammation is closely associated with obesity and related metabolic disorders. However, its origin during obesity is largely unknown. Here, we report that ubiquitin-conjugating enzyme E2M (UBE2M) is critical to obesity-related inflammation induced by macrophages. In mice with UBE2M-deficient macrophages, obesity, insulin resistance, and hepatic steatosis induced by a high-fat diet are greatly alleviated, an effect related to the decreased proinflammatory activity of macrophages due to reduced IL-1β production. Mechanistically, UBE2M deficiency inhibits the neddylation of E3 ubiquitin ligase TRIM21 on K129/134, leading to reduced recruitment and ubiquitination-mediated degradation of E3 ubiquitin ligase VHL. Subsequently, VHL reduces HIF-1α-induced IL-1β production by degrading HIF-1α. Targeting macrophage TRIM21 with Trim21 antisense oligonucleotide-loaded red blood cell extracellular vesicles effectively inhibits obesity-induced inflammation and related metabolic disorders. Thus, our results demonstrate that macrophage UBE2M is essential for obesity-induced inflammation and that TRIM21 is a proof-of-concept target for treating obesity and associated metabolic diseases.

Keywords:  TRIM21; UBE2M; inflammation; neddylation; obesity

DOI:  https://doi.org/10.1016/j.cmet.2023.05.011
J Proteome Res. 2023 Jun 21.

Quantitative Proteomics-Based Substrate Screening Revealed Cyclophilin Stabilization Regulated by Deubiquitinase Ubp7.

Yonghong Wang, Qiuyan Lan, Xinyu Cheng, Yuan Gao, Lei Chang, Ping Xu, Yanchang Li.

  Quantitative proteomics has emerged as a crucial approach to identifying ubiquitinated substrates to investigate the functions of ubiquitination in cells. In this regard, although the substrate screening of certain enzymes in the ubiquitin system has been based on proteome or ubiquitinome level measurements, the direct comparison of these two approaches has not been determined to date. To quantitatively compare the efficiency and effectiveness of substrate screening from the entire proteomics to the ubiquitinomics filter, we used yeast deubiquitinating enzyme, Ubp7, as an example to evaluate it in this study. A total of 112 potential ubiquitinated substrates were identified from the ubiquitinomics level, whereas only 27 regulated substrates were identified from the entire proteomic screening, demonstrating the increased efficiency of ubiquitinomics quantitative analysis. Subsequently, we selected cyclophilin A (Cpr1) protein as an example, which was filtered out at the proteomics level but was a promising candidate according to the ubiquitinomics filter. Additional investigations revealed that Cpr1 possessed a K48-linked ubiquitin chain regulated by Ubp7, which may affect its homeostasis and, consequently, sensitivity to the therapeutic drug cyclosporine (CsA).

Keywords:  K48 chain; cyclophilin A; cyclosporine; deubiquitinating enzyme; stable isotope labeling by amino acids; ubiquitin conjugates

DOI:  https://doi.org/10.1021/acs.jproteome.2c00853
bioRxiv. 2023 Jun 05. pii: 2023.05.31.543183. [Epub ahead of print]

Proteostatic tuning underpins the evolution of novel multicellular traits.

Kristopher Montrose, Dung T Lac, Anthony J Burnetti, Kai Tong, G Ozan Bozdag, Mikaela Hukkanen, William C Ratcliff, Juha Saarikangas.

The evolution of multicellularity paved the way for the origin of complex life on Earth, but little is known about the mechanistic basis of early multicellular evolution. Here, we examine the molecular basis of multicellular adaptation in the Multicellularity Long Term Evolution Experiment (MuLTEE). We demonstrate that cellular elongation, a key adaptation underpinning increased biophysical toughness and organismal size, is convergently driven by downregulation of the chaperone Hsp90. Mechanistically, Hsp90-mediated morphogenesis operates by destabilizing the cyclin-dependent kinase Cdc28, resulting in delayed mitosis and prolonged polarized growth. Reintroduction of Hsp90 expression resulted in shortened cells that formed smaller groups with reduced multicellular fitness. Together, our results show how ancient protein folding systems can be tuned to drive rapid evolution at a new level of biological individuality by revealing novel developmental phenotypes.
One sentence summary: Downregulation of Hsp90 decouples cell cycle progression and growth to drive the evolution of macroscopic multicellularity.

DOI: https://doi.org/10.1101/2023.05.31.543183
J Mol Biol. 2023 Jun 20. pii: S0022-2836(23)00282-6. [Epub ahead of print] 168184

J-domain proteins form binary complexes with Hsp90 and ternary complexes with Hsp90 and Hsp70.

Anushka C Wickramaratne, Jui-Yun Liao, Shannon M Doyle, Joel R Hoskins, Gabrielle Puller, Madison L Scott, John Paul Alao, Ikponwmosa Obaseki, Jerry C Dinan, Tapan K Maity, Lisa M Jenkins, Andrea N Kravats, Sue Wickner.

  Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.

Keywords:  DnaJ; Hsp82; HtpG; Ydj1; molecular chaperones

DOI:  https://doi.org/10.1016/j.jmb.2023.168184
bioRxiv. 2023 Jun 07. pii: 2023.06.05.543769. [Epub ahead of print]

SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.

Erica Wolin, Jimmy K Guo, Mario R Blanco, Andrew A Perez, Isabel N Goronzy, Ahmed A Abdou, Darvesh Gorhe, Mitchell Guttman, Marko Jovanovic.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5'-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

DOI: https://doi.org/10.1101/2023.06.05.543769
Mol Cell Proteomics. 2023 Jun 19. pii: S1535-9476(23)00113-5. [Epub ahead of print] 100602

Proteomic Dynamics of Breast Cancer Cell Lines Identifies Potential Therapeutic Protein Targets.

Rui Sun, Weigang Ge, Yi Zhu, Azin Sayad, Augustin Luna, Mengge Lyu, Shuang Liang, Luis Tobalina, Vinodh N Rajapakse, Chenhuan Yu, Huanhuan Zhang, Jie Fang, Fang Wu, Hui Xie, Julio Saez-Rodriguez, Huazhong Ying, William C Reinhold, Chris Sander, Yves Pommier, Benjamin G Neel, Ruedi Aebersold, Tiannan Guo.

  Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating prior multi-omics datasets with our proteomic results, we found that including proteomics data improved drug sensitivity predictions and provided insights into mechanism of action. We then profiled the proteome changes in nine cell lines (five TNBC, four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provides insights into adaptive resistance in TNBC.

Keywords:  Data-independent acquisition; Proteomics; Proteotype; Triple-negative breast cancer

DOI:  https://doi.org/10.1016/j.mcpro.2023.100602
Nat Commun. 2023 Jun 21. 14(1): 3689

How soluble misfolded proteins bypass chaperones at the molecular level.

Ritaban Halder, Daniel A Nissley, Ian Sitarik, Yang Jiang, Yiyun Rao, Quyen V Vu, Mai Suan Li, Justin Pritchard, Edward P O'Brien.

Subpopulations of soluble, misfolded proteins can bypass chaperones within cells. The extent of this phenomenon and how it happens at the molecular level are unknown. Through a meta-analysis of the experimental literature we find that in all quantitative protein refolding studies there is always a subpopulation of soluble but misfolded protein that does not fold in the presence of one or more chaperones, and can take days or longer to do so. Thus, some misfolded subpopulations commonly bypass chaperones. Using multi-scale simulation models we observe that the misfolded structures that bypass various chaperones can do so because their structures are highly native like, leading to a situation where chaperones do not distinguish between the folded and near-native-misfolded states. More broadly, these results provide a mechanism by which long-time scale changes in protein structure and function can persist in cells because some misfolded states can bypass components of the proteostasis machinery.

DOI: https://doi.org/10.1038/s41467-023-38962-z
Cell Rep. 2023 Jun 21. pii: S2211-1247(23)00677-0. [Epub ahead of print]42(7): 112666

Modulation of cellular metabolism by protein crotonylation regulates pancreatic cancer progression.

Yan Zheng, Le Zhu, Zhao-Yu Qin, Yu Guo, Shun Wang, Min Xue, Ke-Yu Shen, Bei-Yuan Hu, Xu-Feng Wang, Chao-Qun Wang, Lun-Xiu Qin, Qiong-Zhu Dong.

  Protein lysine crotonylation has been recently identified as a vital posttranslational modification in cellular processes, particularly through the modification of histones. We show that lysine crotonylation is an important modification of the cytoplastic and mitochondria proteins. Enzymes in glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid metabolism, glutamine metabolism, glutathione metabolism, the urea cycle, one-carbon metabolism, and mitochondrial fusion/fission dynamics are found to be extensively crotonylated in pancreatic cancer cells. This modulation is mainly controlled by a pair of crotonylation writers and erasers including CBP/p300, HDAC1, and HDAC3. The dynamic crotonylation of metabolic enzymes is involved in metabolism regulation, which is linked with tumor progression. Interestingly, the activation of MTHFD1 by decrotonylation at Lys354 and Lys553 promotes the development of pancreatic cancer by increasing resistance to ferroptosis. Our study suggests that crotonylation represents a metabolic regulatory mechanism in pancreatic cancer progression.

Keywords:  CP: Cancer; CP: Molecular biology; MTHFD1; crotonylation; metabolism; pancreatic cancer; tumor progression

DOI:  https://doi.org/10.1016/j.celrep.2023.112666
Nat Commun. 2023 Jun 23. 14(1): 3755

Mechanistic insights into the aggregation pathway of the patient-derived immunoglobulin light chain variable domain protein FOR005.

Tejaswini Pradhan, Riddhiman Sarkar, Kevin M Meighen-Berger, Matthias J Feige, Martin Zacharias, Bernd Reif.

Systemic antibody light chain (AL) amyloidosis is characterized by deposition of amyloid fibrils. Prior to fibril formation, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes. We focus on the patient derived λ-III AL variable domain FOR005 which is mutated at five positions with respect to the closest germline protein. Using solution-state NMR spectroscopy, we follow the individual steps involved in protein misfolding from the native to the amyloid fibril state. Unfavorable mutations in the complementary determining regions introduce a strain in the native protein structure which yields partial unfolding. Driven by electrostatic interactions, the protein converts into a high molecular weight, oligomeric, molten globule. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into fibrils. The topology is determined by balanced electrostatic interactions in the fibril core implying a 180° rotational switch of the beta-sheets around the conserved disulfide bond.

DOI: https://doi.org/10.1038/s41467-023-39280-0