bims-proteo 2023-02-05 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒02‒05
33 papers selected by
Eric Chevet
INSERM

CCPG1 recognizes ER luminal proteins for selective ER-phagy.
Simultaneous proteome localization and turnover analysis reveals spatiotemporal dynamics of unfolded protein responses.
Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly.
The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48.
A distinct linker mediating ER-autophagosome membrane contact site (EACS) in plants.
Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators.
The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds in a cellular system.
Activity-based profiling of cullin-RING ligase networks by conformation-specific probes.
Phosphorylation of EIF2S1 (eukaryotic translation initiation factor 2 subunit alpha) is indispensable for nuclear translocation of TFEB and TFE3 during ER stress.
The many hats of transmembrane emp24 domain protein TMED9 in secretory pathway homeostasis.
Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the Glucocorticoid Receptor.
Differential requirement for BRCA1-BARD1 E3 ubiquitin ligase activity in DNA damage repair and meiosis in the Caenorhabditis elegans germ line.
Deep-learning based approach to identify substrates of human E3 ubiquitin ligases and deubiquitinases.
The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.
Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.
Deciphering functional roles and interplay between Beclin1 and Beclin2 in autophagosome formation and mitophagy.
O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits.
Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.
Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.
The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5.
Lysosomal quality control: molecular mechanisms and therapeutic implications.
An affinity-directed phosphatase, AdPhosphatase, system for targeted protein dephosphorylation.
FUT2-dependent fucosylation of HYOU1 protects intestinal stem cells against inflammatory injury by regulating unfolded protein response.
PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination.
Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation.
Sample multiplexing-based targeted pathway proteomics with real-time analytics reveals the impact of genetic variation on protein expression.
Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.
Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast.
Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.
Activation Dynamics of Ubiquitin Specific Protease 7.
MANF stimulates autophagy and restores mitochondrial homeostasis to treat toxic proteinopathy.
A genome-wide CRISPR functional survey of the human phagocytosis molecular machinery.
A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell.

Mol Biol Cell. 2023 Feb 03. mbcE22090432

CCPG1 recognizes ER luminal proteins for selective ER-phagy.

Shunsuke Ishii, Haruka Chino, Koji L Ode, Yoshitaka Kurikawa, Hiroki R Ueda, Akira Matsuura, Noboru Mizushima, Eisuke Itakura.

The endoplasmic reticulum (ER) is a major cell compartment where protein synthesis, folding and post-translational modifications occur with assistance from a wide variety of chaperones and enzymes. Quality control systems selectively eliminate abnormal proteins that accumulate inside the ER due to cellular stresses. ER-phagy, i.e., selective autophagy of the ER, is a mechanism that maintains or re-establishes cellular and ER-specific homeostasis through removal of abnormal proteins. However, how ER luminal proteins are recognized by the ER-phagy machinery remains unclear. Here, we applied the aggregation-prone protein, six-repeated islet amyloid polypeptide (6xIAPP), as a model ER-phagy substrate, and found that cell cycle progression 1 (CCPG1), which is an ER-phagy receptor, efficiently mediates its degradation via ER-phagy. We also identified prolyl 3-hydroxylase family member 4 (P3H4) as an endogenous cargo of CCPG1-dependent ER-phagy. The ER luminal region of CCPG1 contains several highly conserved regions that we refer to as cargo interaction regions (CIRs); these directly interact with specific luminal cargos for ER-phagy. Notably, 6xIAPP and P3H4 directly interact with different CIRs. These findings indicate that CCPG1 is a bispecific ER-phagy receptor for ER luminal proteins and the autophagosomal membrane that contributes to the efficient removal of aberrant ER-resident proteins through ER-phagy.

DOI: https://doi.org/10.1091/mbc.E22-09-0432
bioRxiv. 2023 Jan 04. pii: 2023.01.04.521821. [Epub ahead of print]

Simultaneous proteome localization and turnover analysis reveals spatiotemporal dynamics of unfolded protein responses.

Jordan Currie, Vyshnavi Manda, Veronica Hidalgo, R W Ludwig, Maggie P Y Lam, Edward Lau.

The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under protein misfolding stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins, yet a global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, named Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge, we find that the global suppression of protein synthesis during UPR is dependent on subcellular localization, with more severe slowdown in lysosome vs. endoplasmic reticulum (ER) protein turnover. Most candidate translocation events affect pre-existing proteins and likely involve vesicular transport across endomembrane fractions including an expansion of an ER-derived vesicle (ERV) compartment containing RNA binding proteins and stress response proteins. In parallel, we observed specific translocations involving only newly synthesized protein pools that are indicative of endomembrane stalling. The translocation of a subclass of cell surface proteins to the endomembrane including EGFR and ITGAV upon UPR affects only heavy labeled proteins, which suggest their internalization is driven by nascent protein trafficking rather than ligand dependent endocytosis. The approach described here may be broadly useful for inferring the coordinations between spatial and temporal proteome regulations in normal and stressed cells.

DOI: https://doi.org/10.1101/2023.01.04.521821
EMBO J. 2023 Jan 30. e112253

Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly.

Carlos Pla-Prats, Simone Cavadini, Georg Kempf, Nicolas H Thomä.

  Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4-RBX1-DDB1-DCAF12 (CRL4DCAF12 ) E3 ligase preferentially ubiquitinates proteins that carry a C-terminal double glutamate (di-Glu) motif. Reported CRL4DCAF12 di-Glu-containing substrates include CCT5, a subunit of the TRiC chaperonin. How DCAF12 engages its substrates and the functional relationship between CRL4DCAF12 and CCT5/TRiC is currently unknown. Here, we present the cryo-EM structure of the DDB1-DCAF12-CCT5 complex at 2.8 Å resolution. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of the β-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through Van der Waals interactions. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that DCAF12 binds and ubiquitinates monomeric CCT5, but not CCT5 assembled into TRiC. Our biochemical and structural results suggest a previously unknown role for the CRL4DCAF12 E3 ligase in overseeing the assembly of a key cellular complex.

Keywords:  Assembly Quality Control; CCT5; DCAF12; TRiC; Ubiquitin

DOI:  https://doi.org/10.15252/embj.2022112253
Mol Cell. 2023 Jan 25. pii: S1097-2765(23)00039-4. [Epub ahead of print]

The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48.

Cameron Williams, Ken C Dong, Connor Arkinson, Andreas Martin.

  The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.

Keywords:  AAA+ ATPase; Cdc48/p97; Ufd1/Npl4; linkage specificity; ubiquitin-dependent protein unfolding; ubiquitin-proteasome system; unfolding initiation

DOI:  https://doi.org/10.1016/j.molcel.2023.01.016
Autophagy. 2023 Feb 02.

A distinct linker mediating ER-autophagosome membrane contact site (EACS) in plants.

Hao Ye, Jiayang Gao, Liwen Jiang.

  Endoplasmic reticulum (ER) membrane contacts play a central role in regulating autophagosome formation in yeast and mammals. However, a direct functional linkage between the ER and autophagosomes in plants remains elusive. We have recently identified and characterized a plant-unique protein complex consisting of AT4G22540/OSBP2A/ORP2A (oxysterol binding protein-related protein 2A), the ER resident protein AT3G60600/VAP27-1 (vesicle-associated protein 27-1) and AT2G45170/ATG8e (autophagy related 8e) that mediate the ER-autophagosome membrane contact site (EACS) in the model plant Arabidopsis thaliana. Knockdown (KD) of ORP2A affects autophagosome formation and seedling development, whereas ORP2A KD root cells show accumulation of the macroautophagic/autophagic core machinery and PtdIns3P in enlarged ER membranes under autophagy conditions. This study reveals the molecular architecture and functions of a distinct plant EACS in regulating autophagosome formation via lipid redistribution.

Keywords:  Arabidopsis; ORP; PtdIns3P; autophagosome; membrane contact site

DOI:  https://doi.org/10.1080/15548627.2023.2176028
bioRxiv. 2023 Jan 17. pii: 2023.01.16.524237. [Epub ahead of print]

Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Gabriel M Kline, Ryan J Paxman, Chung-Yon Lin, Nicole Madrazo, Julia M Grandjean, Kyunga Lee, Karina Nugroho, Evan T Powers, R Luke Wiseman, Jeffery W Kelly.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino- p -cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino- p -cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

DOI: https://doi.org/10.1101/2023.01.16.524237
Nat Commun. 2023 Feb 02. 14(1): 560

The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds in a cellular system.

Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W Sanders, Saurabh Gautam, Marc I Diamond, F Ulrich Hartl, Mark S Hipp.

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimer's disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the hexameric ATPase valosin-containing protein (VCP) is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with heat shock 70 kDa protein (Hsp70) and the ubiquitin-proteasome machinery. While inhibition of VCP activity stabilizes large Tau aggregates, disaggregation by VCP generates seeding-active Tau species as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.

DOI: https://doi.org/10.1038/s41467-023-36058-2
bioRxiv. 2023 Jan 17. pii: 2023.01.14.524048. [Epub ahead of print]

Activity-based profiling of cullin-RING ligase networks by conformation-specific probes.

Lukas T Henneberg, Jaspal Singh, David M Duda, Kheewoong Baek, David Yanishevski, Peter J Murray, Matthias Mann, Sachdev S Sidhu, Brenda Schulman.

The cullin-RING E3 ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We report development of a synthetic antibody recognizing the active conformation of a NEDD8-linked cullin. We established a pipeline probing cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing CRLs, revealing the CRL complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, prime efficiency of targeted protein degradation, and are differentially rewired across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate development of degrader drugs.

DOI: https://doi.org/10.1101/2023.01.14.524048
Autophagy. 2023 Jan 31.

Phosphorylation of EIF2S1 (eukaryotic translation initiation factor 2 subunit alpha) is indispensable for nuclear translocation of TFEB and TFE3 during ER stress.

Thao Thi Dang, Mi-Jeong Kim, Yoon Young Lee, Hien Thi Le, Kook Hwan Kim, Somi Nam, Seung Hwa Hyun, Hong Lim Kim, Su Wol Chung, Hun Taeg Chung, Eek-Hoon Jho, Hiderou Yoshida, Kyoungmi Kim, Chan Young Park, Myung-Shik Lee, Sung Hoon Back.

  There are diverse links between macroautophagy/autophagy pathways and unfolded protein response (UPR) pathways under endoplasmic reticulum (ER) stress conditions to restore ER homeostasis. Phosphorylation of EIF2S1/eIF2α is an important mechanism that can regulate all three UPR pathways through transcriptional and translational reprogramming to maintain cellular homeostasis and overcome cellular stresses. In this study, to investigate the roles of EIF2S1 phosphorylation in regulation of autophagy during ER stress, we used EIF2S1 phosphorylation-deficient (A/A) cells in which residue 51 was mutated from serine to alanine. A/A cells exhibited defects in several steps of autophagic processes (such as autophagosome and autolysosome formation) that are regulated by the transcriptional activities of the autophagy master transcription factors TFEB and TFE3 under ER stress conditions. EIF2S1 phosphorylation was required for nuclear translocation of TFEB and TFE3 during ER stress. In addition, EIF2AK3/PERK, PPP3/calcineurin-mediated dephosphorylation of TFEB and TFE3, and YWHA/14-3-3 dissociation were required for their nuclear translocation, but were insufficient to induce their nuclear retention during ER stress. Overexpression of the activated ATF6/ATF6α form, XBP1s, and ATF4 differentially rescued defects of TFEB and TFE3 nuclear translocation in A/A cells during ER stress. Consequently, overexpression of the activated ATF6 or TFEB form more efficiently rescued autophagic defects, although XBP1s and ATF4 also displayed an ability to restore autophagy in A/A cells during ER stress. Our results suggest that EIF2S1 phosphorylation is important for autophagy and UPR pathways, to restore ER homeostasis and reveal how EIF2S1 phosphorylation connects UPR pathways to autophagy.

Keywords:  ATF6; EIF2S1 phosphorylation; ER stress; TFEB; autophagy; nuclear translocation; phosphorylation TFE3; transcription factor E3; transcription factor EB

DOI:  https://doi.org/10.1080/15548627.2023.2173900
Front Cell Dev Biol. 2022 ;10 1096899

The many hats of transmembrane emp24 domain protein TMED9 in secretory pathway homeostasis.

Benjamin S Roberts, Prasanna Satpute-Krishnan.

  The secretory pathway is an intracellular highway for the vesicular transport of newly synthesized proteins that spans the endoplasmic reticulum (ER), Golgi, lysosomes and the cell surface. A variety of cargo receptors, chaperones, and quality control proteins maintain the smooth flow of cargo along this route. Among these is vesicular transport protein TMED9, which belongs to the p24/transmembrane emp24 domain (TMED) family of proteins, and is expressed across vertebrate species. The TMED family is comprised of structurally-related type I transmembrane proteins with a luminal N-terminal Golgi-dynamics domain, a luminal coiled-coil domain, a transmembrane domain and a short cytosolic C-terminal tail that binds COPI and COPII coat proteins. TMED9, like other members of the TMED family, was first identified as an abundant constituent of the COPI and COPII coated vesicles that mediate traffic between the ER and the Golgi. TMED9 is typically purified in hetero-oligomers together with TMED family members, suggesting that it may function as part of a complex. Recently, TMED family members have been discovered to play various roles in secretory pathway homeostasis including secreted protein processing, quality control and degradation of misfolded proteins, and post-Golgi trafficking. In particular, TMED9 has been implicated in autophagy, lysosomal sorting, viral replication and cancer, which we will discuss in this Mini-Review.

Keywords:  COP Coatomer; Golgi; autophagy; cargo receptor; endoplasmic reticulum; p24 family; secretory pathway homeostasis; transmembrane emp24 domain

DOI:  https://doi.org/10.3389/fcell.2022.1096899
bioRxiv. 2023 Jan 16. pii: 2023.01.10.523504. [Epub ahead of print]

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the Glucocorticoid Receptor.

Chari M Noddings, Jill L Johnson, David A Agard.

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR) 1-3 . Previously, we revealed that GR ligand binding activity is inhibited by Hsp70 and restored by Hsp90, aided by co-chaperones 4 . We then presented cryo-EM structures mechanistically detailing how Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding 5,6 . In vivo , GR-chaperone complexes are found associated with numerous Hsp90 co-chaperones, but the most enigmatic have been the immunophilins FKBP51 and FKBP52, which further regulate the activity of GR and other steroid receptors 7-9 . A molecular understanding of how FKBP51 and FKBP52 integrate with the GR chaperone cycle to differentially regulate GR activation in vivo is lacking due to difficulties reconstituting these interactions. Here, we present a 3.01 Å cryo-EM structure of the GR:Hsp90:FKBP52 complex, revealing, for the first time, that FKBP52 directly binds to the folded, ligand-bound GR using three novel interfaces, each of which we demonstrate are critical for FKBP52-dependent potentiation of GR activity in vivo . In addition, we present a 3.23 Å cryo-EM structure of the GR:Hsp90:FKBP51 complex, which, surprisingly, largely mimics the GR:Hsp90:FKBP52 structure. In both structures, FKBP51 and FKBP52 directly engage the folded GR and unexpectedly facilitate release of p23 through an allosteric mechanism. We also reveal that FKBP52, but not FKBP51, potentiates GR ligand binding in vitro , in a manner dependent on FKBP52-specific interactions. Altogether, we reveal how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation and how FKBP51 and FKBP52 compete for GR:Hsp90 binding, leading to functional antagonism.

DOI: https://doi.org/10.1101/2023.01.10.523504
PLoS Genet. 2023 Jan 30. 19(1): e1010457

Differential requirement for BRCA1-BARD1 E3 ubiquitin ligase activity in DNA damage repair and meiosis in the Caenorhabditis elegans germ line.

Qianyan Li, Arshdeep Kaur, Kyoko Okada, Richard J McKenney, JoAnne Engebrecht.

The tumor suppressor BRCA1-BARD1 complex regulates many cellular processes; of critical importance to its tumor suppressor function is its role in genome integrity. Although RING E3 ubiquitin ligase activity is the only known enzymatic activity of the complex, the in vivo requirement for BRCA1-BARD1 E3 ubiquitin ligase activity has been controversial. Here we probe the role of BRCA1-BARD1 E3 ubiquitin ligase activity in vivo using C. elegans. Genetic, cell biological, and biochemical analyses of mutants defective for E3 ligase activity suggest there is both E3 ligase-dependent and independent functions of the complex in the context of DNA damage repair and meiosis. We show that E3 ligase activity is important for nuclear accumulation of the complex and specifically to concentrate at meiotic recombination sites but not at DNA damage sites in proliferating germ cells. While BRCA1 alone is capable of monoubiquitylation, BARD1 is required with BRCA1 to promote polyubiquitylation. We find that the requirement for E3 ligase activity and BARD1 in DNA damage signaling and repair can be partially alleviated by driving the nuclear accumulation and self-association of BRCA1. Our data suggest that in addition to E3 ligase activity, BRCA1 may serve a structural role for DNA damage signaling and repair while BARD1 plays an accessory role to enhance BRCA1 function.

DOI: https://doi.org/10.1371/journal.pgen.1010457
Comput Struct Biotechnol J. 2023 ;21 1014-1021

Deep-learning based approach to identify substrates of human E3 ubiquitin ligases and deubiquitinases.

Yixuan Shu, Yanru Hai, Lihua Cao, Jianmin Wu.

  E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs) play key roles in protein degradation. However, a large number of E3 substrate interactions (ESIs) and DUB substrate interactions (DSIs) remain elusive. Here, we present DeepUSI, a deep learning-based framework to identify ESIs and DSIs using the rich information present in protein sequences. Utilizing the collected golden standard dataset, key hyperparameters in the process of model training, including the ones relevant to data sampling and number of epochs, have been systematically assessed. The performance of DeepUSI was thoroughly evaluated by multiple metrics, based on internal and external validation. Application of DeepUSI to cancer-associated E3 and DUB genes identified a list of druggable substrates with functional implications, warranting further investigation. Together, DeepUSI presents a new framework for predicting substrates of E3 ubiquitin ligases and deubiquitinates.

Keywords:  AUPRC, area under the PR curve; AUROC, area under the ROC curve; CNN, convolutional neutral network; DSI, DUB-substrate interaction; DUB, deubiquitinating enzymes; DUB-substrate interactions; Deep learning; E1, ubiquitin-activating enzymes; E2, ubiquitin-conjugating enzymes; E3, ubiquitin ligases; E3-substrate interactions; ESI, E3-substrate interaction; GSP, gold standard positive dataset; PR, precision recall; Pan-cancer analysis; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; UPS, ubiquitin-proteasome system; Ubiquitin proteasome system; Ubiquitination

DOI:  https://doi.org/10.1016/j.csbj.2023.01.021
bioRxiv. 2023 Jan 18. pii: 2023.01.18.524314. [Epub ahead of print]

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

V Nandana, I W Rathnayaka-Mudiyanselage, N S Muthunayake, A Hatami, C B Mousseau, L A Ortiz-Rodr Guez, J Vaishnav, M Collins, A Gega, K S Mallikaarachchi, H Yassine, A Ghosh, J S Biteen, Y Zhu, M M Champion, W S Childers, J M Schrader.

Bacterial RNP bodies (BR-bodies) are non membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here we define the protein substrates of BR-bodies through enrichment of the bodies followed by liquid chromatography-mass spec (LC-MS) proteomic analysis. We found 111 BR-body enriched proteins, including many RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, suggesting BR-bodies are more complex than previously assumed. While most BR-body enriched proteins cannot phase separate, we identified five which can undergo RNA dependent phase separation in vitro , suggesting other RNP condensates interface with BR-bodies. The RNA degradosome protein clients are recruited more strongly to RNase E droplets than that of the other RNP condensates, suggesting that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble unidirectionally, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body associated proteins have the capacity to dissolve the condensate. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro , explaining the observed dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation while coordinating this mode of organization with RNA processing.Highlights: BR-body proteomics identified 111 proteins enriched in BR-bodies.BR-bodies associate with an interconnected network of RNP condensates.BR-body condensation is modulated by its interaction network.RNA is required for rapid BR-body condensation.

DOI: https://doi.org/10.1101/2023.01.18.524314
Life Sci Alliance. 2023 Apr;pii: e202201607. [Epub ahead of print]6(4):

Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.

Simran Goel, Rosario Oliva, Sadasivam Jeganathan, Verian Bader, Laura J Krause, Simon Kriegler, Isabelle D Stender, Chadwick W Christine, Ken Nakamura, Jan-Erik Hoffmann, Roland Winter, Jörg Tatzelt, Konstanze F Winklhofer.

The NF-κB essential modulator NEMO is the core regulatory component of the inhibitor of κB kinase complex, which is a critical checkpoint in canonical NF-κB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1β, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-1β. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional relevance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is defective in mediating IL-1β-induced NF-κB activation.

DOI: https://doi.org/10.26508/lsa.202201607
Sci Signal. 2023 Jan 31. 16(770): eabo4457

Deciphering functional roles and interplay between Beclin1 and Beclin2 in autophagosome formation and mitophagy.

Justin M Quiles, Rita H Najor, Eileen Gonzalez, Monica Jeung, Wenjing Liang, Sarah M Burbach, Erika A Zumaya, Rachel Y Diao, Mark A Lampert, Åsa B Gustafsson.

The degradation of macromolecules and organelles by the process of autophagy is critical for cellular homeostasis and is often compromised during aging and disease. Beclin1 and Beclin2 are implicated in autophagy induction, and these homologs share a high degree of amino acid sequence similarity but have divergent N-terminal regions. Here, we investigated the functions of the Beclin homologs in regulating autophagy and mitophagy, a specialized form of autophagy that targets mitochondria. Both Beclin homologs contributed to autophagosome formation, but a mechanism of autophagosome formation independent of either Beclin homolog occurred in response to starvation or mitochondrial damage. Mitophagy was compromised only in Beclin1-deficient HeLa cells and mouse embryonic fibroblasts because of defective autophagosomal engulfment of mitochondria, and the function of Beclin1 in mitophagy required the phosphorylation of the conserved Ser15 residue by the kinase Ulk1. Mitochondria-ER-associated membranes (MAMs) are important sites of autophagosome formation during mitophagy, and Beclin1, but not Beclin2 or a Beclin1 mutant that could not be phosphorylated at Ser15, localized to MAMs during mitophagy. Our findings establish a regulatory role for Beclin1 in selective mitophagy by initiating autophagosome formation adjacent to mitochondria, a function facilitated by Ulk1-mediated phosphorylation of Ser15 in its distinct N-terminal region.

DOI: https://doi.org/10.1126/scisignal.abo4457
J Biol Chem. 2023 Jan 30. pii: S0021-9258(23)00095-9. [Epub ahead of print] 102963

O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits.

Sadia Rahmani, Hafsa Ahmed, Osemudiamen Ibazebo, Eden Fussner-Dupas, Warren W Wakarchuk, Costin N Antonescu.

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific cargo destined for internalization, generate membrane curvature, and in many cases undergo scission from the plasma membrane to yield intracellular vesicles. The diversity of functions of cell surface proteins controlled via internalization by CME may suggest that regulation of CCP formation could be effective to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. Here, we examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs are controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

DOI: https://doi.org/10.1016/j.jbc.2023.102963
bioRxiv. 2023 Jan 14. pii: 2023.01.13.523943. [Epub ahead of print]

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.

Michael J Podolsky, Benjamin Kheyfets, Afaq H Beigh, Christopher D Yang, Carlos Lizama Valenzuela, Ritwik Datta, Paul J Wolters, Michael T McManus, Ling Qi, Kamran Atabai.

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, we executed unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discovered a previously unappreciated mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism is dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of SEL1L. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus cell-autonomous, rheostatic collagen clearance is a previously unidentified pathway of tissue homeostasis.

DOI: https://doi.org/10.1101/2023.01.13.523943
PNAS Nexus. 2022 Nov;1(5): pgac232

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Sheeba Jacob, Tia H Turner, Jinyang Cai, Konstantinos V Floros, Ann K Yu, Colin M Coon, Rishabh Khatri, Mohammad A Alzubi, Charles T Jakubik, Ynes M Bouck, Madhavi Puchalapalli, Mayuri Shende, Mikhail G Dozmorov, Sosipatros A Boikos, Bin Hu, J Chuck Harrell, Cyril H Benes, Jennifer E Koblinski, Carlotta Costa, Anthony C Faber.

  Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Keywords:  CRISPR; ER stress; UBA1; c-MYC; targeted therapies

DOI:  https://doi.org/10.1093/pnasnexus/pgac232
Elife. 2023 Feb 03. pii: e79013. [Epub ahead of print]12

The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5.

Ryota Miyashita, Atsuya Nishiyama, Weihua Qin, Yoshie Chiba, Satomi Kori, Norie Kato, Chieko Konishi, Soichiro Kumamoto, Hiroko Kozuka-Hata, Masaaki Oyama, Yoshitaka Kawasoe, Toshiki Tsurimoto, Tatsuro S Takahashi, Heinrich Leonhardt, Kyohei Arita, Makoto Nakanishi.

  UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.

Keywords:  cell biology; xenopus

DOI:  https://doi.org/10.7554/eLife.79013
Trends Cell Biol. 2023 Jan 28. pii: S0962-8924(23)00004-1. [Epub ahead of print]

Lysosomal quality control: molecular mechanisms and therapeutic implications.

Haoxiang Yang, Jay Xiaojun Tan.

  Lysosomes are essential catabolic organelles with an acidic lumen and dozens of hydrolytic enzymes. The detrimental consequences of lysosomal leakage have been well known since lysosomes were discovered during the 1950s. However, detailed knowledge of lysosomal quality control mechanisms has only emerged relatively recently. It is now clear that lysosomal leakage triggers multiple lysosomal quality control pathways that replace, remove, or directly repair damaged lysosomes. Here, we review how lysosomal damage is sensed and resolved in mammalian cells, with a focus on the molecular mechanisms underlying different lysosomal quality control pathways. We also discuss the clinical implications and therapeutic potential of these pathways.

Keywords:  ESCRT; PITT; TFEB/TFE3; lysophagy; lysosomal biogenesis; lysosomal repair

DOI:  https://doi.org/10.1016/j.tcb.2023.01.001
Cell Chem Biol. 2023 Jan 25. pii: S2451-9456(23)00026-0. [Epub ahead of print]

An affinity-directed phosphatase, AdPhosphatase, system for targeted protein dephosphorylation.

Luke M Simpson, Luke J Fulcher, Gajanan Sathe, Abigail Brewer, Jin-Feng Zhao, Daniel R Squair, Jennifer Crooks, Melanie Wightman, Nicola T Wood, Robert Gourlay, Joby Varghese, Renata F Soares, Gopal P Sapkota.

  Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signaling. Failure of phospho-control accounts for many human diseases. While a kinase phosphorylates multiple substrates, a substrate is often phosphorylated by multiple kinases. This renders phospho-control at the substrate level challenging, as it requires inhibition of multiple kinases, which would thus affect other kinase substrates. Here, we describe the development and application of the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-substrates. By deploying the Protein Phosphatase 1 or 2A catalytic subunits conjugated to an antigen-stabilized anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags using CRISPR-Cas9, with exquisite specificity. By redirecting protein phosphatases to neo-substrates through nanobody-mediated proximity, AdPhosphatase can alter the phospho-status and function of target proteins and thus, offers a new modality for potential drug discovery approaches.

Keywords:  AdPhosphatase; FAM83D; PPP1CA; PPP2CA; ULK1; affinity-directed phosphatase; nanobody; targeted dephosphorylation

DOI:  https://doi.org/10.1016/j.chembiol.2023.01.003
Redox Biol. 2023 Jan 28. pii: S2213-2317(23)00019-8. [Epub ahead of print]60 102618

FUT2-dependent fucosylation of HYOU1 protects intestinal stem cells against inflammatory injury by regulating unfolded protein response.

Zhe Wang, Chen Tan, Caihan Duan, Junhao Wu, Dan Zhou, Lingzhi Hou, Wei Qian, Chaoqun Han, Xiaohua Hou.

  The intestinal epithelial repair after injury is coordinated by intestinal stem cells (ISCs). Fucosylation catalyzed by fucosyltransferase 2 (FUT2) of the intestinal epithelium is beneficial to mucosal healing but poorly defined is the influence on ISCs. The dextran sulfate sodium (DSS) and lipopolysaccharide (LPS) model were used to assess the role of FUT2 on ISCs after injury. The apoptosis, function, and stemness of ISCs were analyzed using intestinal organoids from WT and Fut2ΔISC (ISC-specific Fut2 knockout) mice incubated with LPS and fucose. N-glycoproteomics, UEA-1 chromatography, and site-directed mutagenesis were monitored to dissect the regulatory mechanism, identify the target fucosylated protein and the corresponding modification site. Fucose could alleviate intestinal epithelial damage via upregulating FUT2 and α-1,2-fucosylation of ISCs. Oxidative stress, mitochondrial dysfunction, and cell apoptosis were impeded by fucose. Meanwhile, fucose sustained the growth and proliferation capacity of intestinal organoids treated with LPS. Contrarily, FUT2 depletion in ISCs aggravated the epithelial damage and disrupted the growth and proliferation capacity of ISCs via escalating LPS-induced endoplasmic reticulum (ER) stress and initiating the IRE1/TRAF2/ASK1/JNK branch of unfolded protein response (UPR). Fucosylation of the chaperone protein HYOU1 at the N-glycosylation site of asparagine (Asn) 862 mediated by FUT2 was identified to facilitate ISCs survival and self-renewal, and improve ISCs resistance to ER stress and inflammatory injury. Our study highlights a fucosylation-dependent protective mechanism of ISCs against inflammation, which may provide a fascinating strategy for treating intestinal injury disorders.

Keywords:  Apoptotsis; FUT2; Fucosylation; Inflammatory injury; Intestinal stem cell; Unfolded protein response

DOI:  https://doi.org/10.1016/j.redox.2023.102618
J Clin Invest. 2023 Feb 01. pii: e160456. [Epub ahead of print]133(3):

PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination.

Xinfang Yu, Wei Li, Haidan Liu, Xu Wang, Cristian Coarfa, Chao Cheng, Xinlian Yu, Zhaoyang Zeng, Ya Cao, Ken H Young, Yong Li.

  Programmed death-ligand 1 (PD-L1), a critical immune checkpoint ligand, is a transmembrane protein synthesized in the endoplasmic reticulum of tumor cells and transported to the plasma membrane to interact with programmed death 1 (PD-1) expressed on T cell surface. This interaction delivers coinhibitory signals to T cells, thereby suppressing their function and allowing evasion of antitumor immunity. Most companion or complementary diagnostic devices for assessing PD-L1 expression levels in tumor cells used in the clinic or in clinical trials require membranous staining. However, the mechanism driving PD-L1 translocation to the plasma membrane after de novo synthesis is poorly understood. Herein, we showed that mind bomb homolog 2 (MIB2) is required for PD-L1 transportation from the trans-Golgi network (TGN) to the plasma membrane of cancer cells. MIB2 deficiency led to fewer PD-L1 proteins on the tumor cell surface and promoted antitumor immunity in mice. Mechanistically, MIB2 catalyzed nonproteolytic K63-linked ubiquitination of PD-L1, facilitating PD-L1 trafficking through Ras-associated binding 8-mediated (RAB8-mediated) exocytosis from the TGN to the plasma membrane, where it bound PD-1 extrinsically to prevent tumor cell killing by T cells. Our findings demonstrate that nonproteolytic ubiquitination of PD-L1 by MIB2 is required for its transportation to the plasma membrane and tumor cell immune evasion.

Keywords:  Cancer; Cancer immunotherapy; Cell Biology; Cellular immune response; Oncology

DOI:  https://doi.org/10.1172/JCI160456
Commun Biol. 2023 Jan 30. 6(1): 121

Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation.

Jonathan D Hibshman, Serena Carra, Bob Goldstein.

Small heat shock proteins (sHSPs) are chaperones with well-characterized roles in heat stress, but potential roles for sHSPs in desiccation tolerance have not been as thoroughly explored. We identified nine sHSPs from the tardigrade Hypsibius exemplaris, each containing a conserved alpha-crystallin domain flanked by disordered regions. Many of these sHSPs are highly expressed. Multiple tardigrade and human sHSPs could improve desiccation tolerance of E. coli, suggesting that the capacity to contribute to desicco-protection is a conserved property of some sHSPs. Purification and subsequent analysis of two tardigrade sHSPs, HSP21 and HSP24.6, revealed that these proteins can oligomerize in vitro. These proteins limited heat-induced aggregation of the model enzyme citrate synthase. Heterologous expression of HSP24.6 improved bacterial heat shock survival, and the protein significantly reduced heat-induced aggregation of soluble bacterial protein. Thus, HSP24.6 likely chaperones against protein aggregation to promote heat tolerance. Furthermore, HSP21 and HSP24.6 limited desiccation-induced aggregation and loss of function of citrate synthase. This suggests a mechanism by which tardigrade sHSPs promote desiccation tolerance, by limiting desiccation-induced protein aggregation, thereby maintaining proteostasis and supporting survival. These results suggest that sHSPs provide a mechanism of general stress resistance that can also be deployed to support survival during anhydrobiosis.

DOI: https://doi.org/10.1038/s42003-023-04512-y
Nat Commun. 2023 Feb 02. 14(1): 555

Sample multiplexing-based targeted pathway proteomics with real-time analytics reveals the impact of genetic variation on protein expression.

Qing Yu, Xinyue Liu, Mark P Keller, Jose Navarrete-Perea, Tian Zhang, Sipei Fu, Laura P Vaites, Steven R Shuken, Ernst Schmid, Gregory R Keele, Jiaming Li, Edward L Huttlin, Edrees H Rashan, Judith Simcox, Gary A Churchill, Devin K Schweppe, Alan D Attie, Joao A Paulo, Steven P Gygi.

Targeted proteomics enables hypothesis-driven research by measuring the cellular expression of protein cohorts related by function, disease, or class after perturbation. Here, we present a pathway-centric approach and an assay builder resource for targeting entire pathways of up to 200 proteins selected from >10,000 expressed proteins to directly measure their abundances, exploiting sample multiplexing to increase throughput by 16-fold. The strategy, termed GoDig, requires only a single-shot LC-MS analysis, ~1 µg combined peptide material, a list of up to 200 proteins, and real-time analytics to trigger simultaneous quantification of up to 16 samples for hundreds of analytes. We apply GoDig to quantify the impact of genetic variation on protein expression in mice fed a high-fat diet. We create several GoDig assays to quantify the expression of multiple protein families (kinases, lipid metabolism- and lipid droplet-associated proteins) across 480 fully-genotyped Diversity Outbred mice, revealing protein quantitative trait loci and establishing potential linkages between specific proteins and lipid homeostasis.

DOI: https://doi.org/10.1038/s41467-023-36269-7
bioRxiv. 2023 Jan 05. pii: 2023.01.05.522830. [Epub ahead of print]

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Anil Kumar Vijjamarri, Xiao Niu, Matthew D Vandermeulen, Chisom Onu, Fan Zhang, Hongfang Qiu, Neha Gupta, Swati Gaikwad, Miriam L Greenberg, Paul J Cullen, Zhenguo Lin, Alan G Hinnebusch.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2 Δ cells that appears to result directly from impaired decapping rather than elevated transcription, which was confirmed by ChIP-Seq analysis of RNA Polymerase II occupancies genome-wide. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Lsm2, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2 Δ confers widespread changes in relative TEs that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2 Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2 Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are derepressed, and both mitochondrial function and cell filamentation (a strategy for nutrient foraging) are elevated by dcp2 Δ, suggesting that mRNA decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

DOI: https://doi.org/10.1101/2023.01.05.522830
Mol Cell Biol. 2023 Jan;43(1): 64-84

Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast.

Marjan Moallem, Akhi Akhter, Giovanni L Burke, John Babu, Benjamin G Bergey, J Bryan McNeil, Mohammad S Baig, Emanuel Rosonina.

  Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.

Keywords:  Ubc9; Ulp1; heat shock; sumoylation; transcription; yeast

DOI:  https://doi.org/10.1080/10985549.2023.2166320
Mol Cell. 2023 Jan 31. pii: S1097-2765(23)00038-2. [Epub ahead of print]

Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.

Martin Golkowski, Andrea Lius, Tanmay Sapre, Ho-Tak Lau, Taylor Moreno, Dustin J Maly, Shao-En Ong.

  Dynamic changes in protein-protein interaction (PPI) networks underlie all physiological cellular functions and drive devastating human diseases. Profiling PPI networks can, therefore, provide critical insight into disease mechanisms and identify new drug targets. Kinases are regulatory nodes in many PPI networks; yet, facile methods to systematically study kinase interactome dynamics are lacking. We describe kinobead competition and correlation analysis (kiCCA), a quantitative mass spectrometry-based chemoproteomic method for rapid and highly multiplexed profiling of endogenous kinase interactomes. Using kiCCA, we identified 1,154 PPIs of 238 kinases across 18 diverse cancer lines, quantifying context-dependent kinase interactome changes linked to cancer type, plasticity, and signaling states, thereby assembling an extensive knowledgebase for cell signaling research. We discovered drug target candidates, including an endocytic adapter-associated kinase (AAK1) complex that promotes cancer cell epithelial-mesenchymal plasticity and drug resistance. Our data demonstrate the importance of kinase interactome dynamics for cellular signaling in health and disease.

Keywords:  kinome; native complex; protein interactions; proteomics; signaling pathways

DOI:  https://doi.org/10.1016/j.molcel.2023.01.015
bioRxiv. 2023 Jan 12. pii: 2023.01.11.523550. [Epub ahead of print]

Activation Dynamics of Ubiquitin Specific Protease 7.

Gabrielle J Valles, Nancy Jaiswal, Dmitry M Korzhnev, Irina Bezsonova.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its "invisible" active conformation in solution, which may contribute to its activation mechanism.

DOI: https://doi.org/10.1101/2023.01.11.523550
bioRxiv. 2023 Jan 12. pii: 2023.01.10.523171. [Epub ahead of print]

MANF stimulates autophagy and restores mitochondrial homeostasis to treat toxic proteinopathy.

Yeawon Kim, Chuang Li, Chenjian Gu, Eric Tycksen, Anuradhika Puri, Terri A Pietka, Jothilingam Sivapackiam, Yili Fang, Kendrah Kidd, Sun-Ji Park, Bryce G Johnson, Stanislav Kmoch, Jeremy S Duffield, Anthony J Bleyer, Meredith E Jackrel, Fumihiko Urano, Vijay Sharma, Maria Lindahl, Ying Maggie Chen.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD- UMOD ), one of the leading hereditary kidney diseases, and Alzheimerâ€™s disease etc. There are no targeted therapies. ADTKD is also a genetic form of renal fibrosis and chronic kidney disease, which affects 500 million people worldwide. For the first time, in our newly generated mouse model recapitulating human ADTKD- UMOD carrying a leading UMOD deletion mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are severely impaired, leading to cGAS- STING activation and tubular injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel endoplasmic reticulum stress-regulated secreted protein. We provide the first study that inducible tubular overexpression of MANF after the onset of disease stimulates autophagy/mitophagy and clearance of the misfolded UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, resulting in protection of kidney function. Conversely, genetic ablation of endogenous MANF upregulated in the mutant mouse and human tubular cells worsens autophagy suppression and kidney fibrosis. Together, we discover MANF as a novel biotherapeutic protein and elucidate previously unknown mechanisms of MANF in regulating organelle homeostasis to treat ADTKD, which may have broad therapeutic application to treat various proteinopathies.

DOI: https://doi.org/10.1101/2023.01.10.523171
Life Sci Alliance. 2023 Apr;pii: e202201715. [Epub ahead of print]6(4):

A genome-wide CRISPR functional survey of the human phagocytosis molecular machinery.

Patrick Essletzbichler, Vitaly Sedlyarov, Fabian Frommelt, Didier Soulat, Leonhard X Heinz, Adrijana Stefanovic, Benedikt Neumayer, Giulio Superti-Furga.

Phagocytosis, the process by which cells engulf large particles, plays a vital role in driving tissue clearance and host defense. Its dysregulation is connected to autoimmunity, toxic accumulation of proteins, and increased risks for infections. Despite its importance, we lack full understanding of all molecular components involved in the process. To create a functional map in human cells, we performed a genome-wide CRISPRko FACS screen that identified 716 genes. Mapping those hits to a comprehensive protein-protein interaction network annotated for functional cellular processes allowed retrieval of protein complexes identified multiple times and detection of missing phagocytosis regulators. In addition to known components, such as the Arp2/3 complex, the vacuolar-ATPase-Rag machinery, and the Wave-2 complex, we identified and validated new phagocytosis-relevant functions, including the oligosaccharyltransferase complex (MAGT1/SLC58A1, DDOST, STT3B, and RPN2) and the hypusine pathway (eIF5A, DHPS, and DOHH). Overall, our phagocytosis network comprises elements of cargo uptake, shuffling, and biotransformation through the cell, providing a resource for the identification of potential novel drivers for diseases of the endo-lysosomal system. Our approach of integrating protein-protein interaction offers a broadly applicable way to functionally interpret genome-wide screens.

DOI: https://doi.org/10.26508/lsa.202201715
Nucleic Acids Res. 2023 Jan 30. pii: gkac1276. [Epub ahead of print]

A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell.

Ayako Yamakawa, Tatsuya Niwa, Yuhei Chadani, Akinao Kobo, Hideki Taguchi.

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains, i.e. polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods to investigate nascent chains in the cell.

DOI: https://doi.org/10.1093/nar/gkac1276