bims-proteo 2023-11-05 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒11‒05
thirty-one papers selected by
Eric Chevet, INSERM

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress.
TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.
How degrading! Trapped translation factors get trashed.
Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation.
Autophagy of the ER: the secretome finds the lysosome.
The E3 ubiquitin ligase RNF216 contains a linear ubiquitin chain-determining-like domain that functions to regulate dendritic arborization and dendritic spine type in hippocampal neurons.
Loss of Grp170 results in catastrophic disruption of endoplasmic reticulum functions.
UVRAG cooperates with cargo receptors to assemble the ER-phagy site.
Thiol reductive stress activates the hypoxia response pathway.
Cotranslational sorting and processing of newly synthesized proteins in eukaryotes.
Septins modulate the autophagy response after nutrient starvation.
Chondrodysplasia-inducing COL2A1 p.Gly1170Ser causes an ER storage defect without associated unfolded protein response in a human cartilage model.
BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1.
Control of ATG14 solubility and autophagy by MARCHF7/MARCH7-mediated ubiquitination.
Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.
SMURF1 controls the PPP3/calcineurin complex and TFEB at a regulatory node for lysosomal biogenesis.
DNA-encoded library-enabled discovery of proximity-inducing small molecules.
Recognition and reprogramming of E3 ubiquitin ligase surfaces by α-helical peptides.
Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes.
Kinetic Modeling of PROTAC-Induced Protein Degradation.
Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics.
Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline.
SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.
The mechanism of Atg15-mediated membrane disruption in autophagy.
Crosstalk between protein misfolding and endoplasmic reticulum stress during ageing and their role in age-related disorders.
CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses.
Structural surfaceomics reveals an AML-specific conformation of integrin β2 as a CAR T cellular therapy target.
Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells.
Shift of the insoluble content of the proteome in the aging mouse brain.
What strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.
Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs.

Cell Rep. 2023 Nov 01. pii: S2211-1247(23)01371-2. [Epub ahead of print]42(11): 113359

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress.

Sezen Meydan, Géssica C Barros, Vanessa Simões, Lana Harley, Blanche K Cizubu, Nicholas R Guydosh, Gustavo M Silva.

  Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation, however, is unclear. We therefore perform Ribo-seq and Disome-seq in Saccharomyces cerevisiae and show that oxidative stress causes ribosome pausing at specific amino acid motifs, which also leads to ribosome collisions. However, these redox-pausing signatures are lost in the absence of Rad6 and do not depend on the ribosome-associated quality control (RQC) pathway. We also show that Rad6 is needed to inhibit overall translation in response to oxidative stress and that its deletion leads to increased expression of antioxidant genes. Finally, we observe that the lack of Rad6 leads to changes during translation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.

Keywords:  CP: Molecular biology; Rad6; oxidative stress; ribosome pause; ribosome ubiquitination; stress response; translation control

DOI:  https://doi.org/10.1016/j.celrep.2023.113359
bioRxiv. 2023 Oct 20. pii: 2023.10.19.563195. [Epub ahead of print]

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Bhaskar Saha, Hallvard Olsvik, Geneva L Williams, Seeun Oh, Gry Evjen, Eva Sjøttem, Michael A Mandell.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α. Mitochondrial damage triggers TRIM5α's auto-ubiquitination and its interaction with ubiquitin-binding autophagy adaptors including NDP52, optineurin, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1. TRIM5α with intact ubiquitination function is required for the proper accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Additionally, we show that TRIM5α can directly recruit autophagy initiation machinery to damaged mitochondria. Our data support a model in which TRIM5α provides a self-amplifying, mitochondria-localized, ubiquitin-based, assembly platform for TBK1 and mitophagy adaptors that is ultimately required to recruit the core autophagy machinery.

DOI: https://doi.org/10.1101/2023.10.19.563195
Cell Rep. 2023 Oct 31. pii: S2211-1247(23)01290-1. [Epub ahead of print]42(11): 113278

How degrading! Trapped translation factors get trashed.

Pierce W Ford, Eric J Bennett.

Using small molecules that trap translation factors within translating ribosomes, Gurzeler et al.1 and Oltion et al.2 identify a new branch of the ribosome-associated quality-control (RQC) pathway. This mode of translation regulation expands the number of mechanistically distinct RQC pathways.

DOI: https://doi.org/10.1016/j.celrep.2023.113278
bioRxiv. 2023 Oct 21. pii: 2023.10.20.563371. [Epub ahead of print]

Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation.

Seong Ho Hong, Akane Osa, Ingrid E Wertz, Daniel K Nomura.

Targeted protein degradation with Proteolysis Targeting Chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.

DOI: https://doi.org/10.1101/2023.10.20.563371
FEBS J. 2023 Nov 03.

Autophagy of the ER: the secretome finds the lysosome.

Jeffrey Knupp, Madison L Pletan, Peter Arvan, Billy Tsai.

  Lysosomal degradation of the endoplasmic reticulum (ER) and its components through the autophagy pathway has emerged as a major regulator of ER proteostasis. Commonly referred to as ER-phagy and ER-to-lysosome-associated degradation (ERLAD), how the ER is targeted to the lysosome has been recently clarified by a growing number of studies. Here, we summarize the discoveries of the molecular components required for lysosomal degradation of the ER and their proposed mechanisms of action. Additionally, we discuss how cells employ these machineries to create the different routes of ER-lysosome-associated degradation. Further, we review the role of ER-phagy in viral infection pathways, as well as the implication of ER-phagy in human disease. In sum, we provide a comprehensive overview of the current field of ER-phagy.

Keywords:  ER-phagy; autophagy; endoplasmic reticulum; protein quality control; virus-host interactions

DOI:  https://doi.org/10.1111/febs.16986
bioRxiv. 2023 Oct 19. pii: 2023.10.19.563080. [Epub ahead of print]

The E3 ubiquitin ligase RNF216 contains a linear ubiquitin chain-determining-like domain that functions to regulate dendritic arborization and dendritic spine type in hippocampal neurons.

Jayashree Kadirvelu, Savannah E Jacobs, Ruochuan Liu, Antoinette J Charles, Jun Yin, Angela M Mabb.

Of the hundreds of E3 ligases found in the human genome, the RING-between RING (RBR) E3 ligase in the LUBAC (linear ubiquitin chain assembly complex) complex HOIP (HOIL-1-interacting protein or RNF31), contains a unique domain called LDD (linear ubiquitin chain determining domain). HOIP is the only E3 ligase known to form linear ubiquitin chains, which regulate inflammatory responses and cell death via activation of the NF-κB pathway. We identified an amino acid sequence within the RNF216 E3 ligase that shares homology to the LDD domain found in HOIP (R2-C). Here, we show that the R2-C domain of RNF216 promotes self-assembly of all ubiquitin chains, with a dominance for those assembled via K63-linkages. Deletion of the R2-C domain altered RNF216 localization, reduced dendritic complexity and changed the distribution of apical dendritic spine morphology types in primary hippocampal neurons. These changes were independent of the RNF216 RBR catalytic activity as expression of a catalytic inactive version of RNF216 had no effect. These data show that the R2-C domain of RNF216 diverges in ubiquitin assembly function from the LDD of HOIP and and functions independently of RNF216 catalytic activity to regulate dendrite development in neurons.

DOI: https://doi.org/10.1101/2023.10.19.563080
bioRxiv. 2023 Oct 20. pii: 2023.10.19.563191. [Epub ahead of print]

Loss of Grp170 results in catastrophic disruption of endoplasmic reticulum functions.

Melissa J Mann, Chris Melendez-Suchi, Maria Sukhoplyasova, Ashley R Flory, Mary Carson Irvine, Anuradha R Iyer, Hannah Vorndran, Christopher J Guerriero, Jeffrey L Brodsky, Linda M Hendershot, Teresa M Buck.

GRP170, a product of the Hyou1 gene, is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds non-native proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of embryonic fibroblasts from mice in which LoxP sites were engineered in the Hyou1 loci ( Hyou1 LoxP/LoxP ). A doxycycline-regulated Cre recombinase was also stably introduced into these cells. Induction of Cre resulted in excision of Hyou1 and depletion of Grp170 protein, culminating in apoptotic cell death. As Grp170 levels fell we observed increased steady-state binding of BiP to a client, slowed degradation of a misfolded BiP substrate, and BiP accumulation in NP40-insoluble fractions. Consistent with disrupted BiP functions, we observed reactivation of BiP storage pools and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and insights into mutations in the Hyou1 locus and human disease.

DOI: https://doi.org/10.1101/2023.10.19.563191
EMBO J. 2023 Oct 30. e113625

UVRAG cooperates with cargo receptors to assemble the ER-phagy site.

Xuehong Qian, Lingang He, Jiejie Yang, Jiajia Sun, Xueying Peng, Yuting Zhang, Yizhou Mao, Ying Zhang, Yixian Cui.

  ER-phagy is a selective autophagy process that targets specific regions of the endoplasmic reticulum (ER) for removal via lysosomal degradation. During cellular stress induced by starvation, cargo receptors concentrate at distinct ER-phagy sites (ERPHS) to recruit core autophagy proteins and initiate ER-phagy. However, the molecular mechanism responsible for ERPHS formation remains unclear. In our study, we discovered that the autophagy regulator UV radiation Resistance-Associated Gene (UVRAG) plays a crucial role in orchestrating the assembly of ERPHS. Upon starvation, UVRAG localizes to ERPHS and interacts with specific ER-phagy cargo receptors, such as FAM134B, ATL3, and RTN3L. UVRAG regulates the oligomerization of cargo receptors and facilitates the recruitment of Atg8 family proteins. Consequently, UVRAG promotes efficient ERPHS assembly and turnover of both ER sheets and tubules. Importantly, UVRAG-mediated ER-phagy contributes to the clearance of pathogenic proinsulin aggregates. Remarkably, the involvement of UVRAG in ER-phagy initiation is independent of its canonical function as a subunit of class III phosphatidylinositol 3-kinase complex II.

Keywords:  Atg8 family protein; Beclin-1; ER-phagy; UVRAG; cargo receptor

DOI:  https://doi.org/10.15252/embj.2023113625
EMBO J. 2023 Oct 02. e114093

Thiol reductive stress activates the hypoxia response pathway.

Ravi, Ajay Kumar, Shalmoli Bhattacharyya, Jogender Singh.

  Owing to their capability to disrupt the oxidative protein folding environment in the endoplasmic reticulum (ER), thiol antioxidants, such as dithiothreitol (DTT), are used as ER-specific stressors. We recently showed that thiol antioxidants modulate the methionine-homocysteine cycle by upregulating an S-adenosylmethionine-dependent methyltransferase, rips-1, in Caenorhabditis elegans. However, the changes in cellular physiology induced by thiol stress that modulate the methionine-homocysteine cycle remain uncharacterized. Here, using forward genetic screens in C. elegans, we discover that thiol stress enhances rips-1 expression via the hypoxia response pathway. We demonstrate that thiol stress activates the hypoxia response pathway. The activation of the hypoxia response pathway by thiol stress is conserved in human cells. The hypoxia response pathway enhances thiol toxicity via rips-1 expression and confers protection against thiol toxicity via rips-1-independent mechanisms. Finally, we show that DTT might activate the hypoxia response pathway by producing hydrogen sulfide. Our studies reveal an intriguing interaction between thiol-mediated reductive stress and the hypoxia response pathway and challenge the current model that thiol antioxidant DTT disrupts only the ER milieu in the cell.

Keywords:  C. elegans; endoplasmic reticulum; hif‐1; hypoxia; reductive stress

DOI:  https://doi.org/10.15252/embj.2023114093
Trends Biochem Sci. 2023 Nov 01. pii: S0968-0004(23)00258-X. [Epub ahead of print]

Cotranslational sorting and processing of newly synthesized proteins in eukaryotes.

Martin Gamerdinger, Elke Deuerling.

  Ribosomes interact with a variety of different protein biogenesis factors that guide newly synthesized proteins to their native 3D shapes and cellular localization. Depending on the type of translated substrate, a distinct set of cotranslational factors must interact with the ribosome in a timely and coordinated manner to ensure proper protein biogenesis. While cytonuclear proteins require cotranslational maturation and folding factors, secretory proteins must be maintained in an unfolded state and processed cotranslationally by transport and membrane translocation factors. Here we explore the specific cotranslational processing steps for cytonuclear, secretory, and membrane proteins in eukaryotes and then discuss how the nascent polypeptide-associated complex (NAC) cotranslationally sorts these proteins into the correct protein biogenesis pathway.

Keywords:  N-acetyltransferase; methionine aminopeptidase; nascent polypeptide-associated complex; ribosome-associated complex; signal recognition particle; translocon Sec61

DOI:  https://doi.org/10.1016/j.tibs.2023.10.003
Mol Biol Cell. 2023 Nov 01. mbcE22110520

Septins modulate the autophagy response after nutrient starvation.

Luis Perucho-Jaimes, Jonathan Do, Alexandria Van Elgort, Kenneth B Kaplan.

The pathways that induce macroautophagy (referred to as autophagy hereafter) in response to the stress of starvation are well conserved and essential under nutrient limiting conditions. However, less is understood about the mechanisms that modulate the autophagy response. Here we present evidence that after induction of autophagy in budding yeast septin filaments rapidly assemble into discrete patches distributed along the cell cortex. These patches gradually mature over 12h of nutrient deprivation to form extended structures around Atg9-membranes tethered at the cortical endoplasmic reticulum, a class of membranes that are limiting for autophagosome biogenesis. Loss of cortical septin structures alters the kinetics of autophagy activation and most dramatically extends the duration of the autophagy response. In wild type cells, diffusion of Atg9-membranes at the cell cortex undergoes transient pauses that are dependent on septins, and septins at the bud neck block the diffusion of Atg9-membranes between mother and daughter cells. We conclude that septins reorganize at the cell cortex during autophagy to locally limit access of Atg9-membranes to autophagosome assembly sites, and thus modulate the autophagy response during nutrient-deprivation. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E22-11-0520
bioRxiv. 2023 Oct 20. pii: 2023.10.19.562780. [Epub ahead of print]

Chondrodysplasia-inducing COL2A1 p.Gly1170Ser causes an ER storage defect without associated unfolded protein response in a human cartilage model.

Kathryn M Yammine, Sophia Mirda Abularach, Seo-Yeon Kim, Agata A Bikovtseva, Jinia Lilianty, Vincent L Butty, Richard P Schiavoni, John F Bateman, Shireen R Lamandé, Matthew D Shoulders.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II ( COL2A1 ), the primary collagen in cartilage, can lead to chondrodysplasias of various severities. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis and Legg-Calvé-Perthes disease. Here, we develop and characterize a novel induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. Biochemical characterization reveals that Gly1170Ser procollagen-II is notably slow to fold and secrete. Procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Intriguingly, perhaps due to the pathologic substitution occurring within a triple-helical domain that lacks hydrophobic character, this ER protein accumulation is not recognized by cellular stress responses, such as the unfolded protein response. Interactome studies show that Gly1170Ser procollagen-II interacts to a greater extent with certain ER chaperones and modifying enzymes than wild-type, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the cartilage model developed here provides a valuable platform to rapidly screen and develop therapeutic strategies that can restore procollagen folding and secretion in this collagenopathy and others.

DOI: https://doi.org/10.1101/2023.10.19.562780
Autophagy. 2023 Oct 30.

BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1.

Heng Lin, Sarah Sandkuhler, Colleen Dunlea, Joel Rodwell-Bullock, Darron H King, Gail V W Johnson.

  Macroautophagy/autophagy receptors are essential for the recognition and clearance of specific cargos by selective autophagy, which is essential for maintaining MAPT proteostasis. Previous studies have implicated different autophagy receptors in directing distinct species of MAPT to autophagy, but the underlying mechanisms have not been fully investigated. Here we examine how the autophagy receptors NBR1 and SQSTM1 differentially associate with specific forms of MAPT. In primary neurons depletion of NBR1, unlike depletion of SQSTM1, significantly increased phosphorylated MAPT levels. The specificity of the interactions was confirmed using in vitro binding assays with purified proteins. We provide direct evidence that the co-chaperone BAG3 promotes the preferential association of NBR1 with monomeric MAPT and SQSTM1 with oligomeric MAPT. Using an in vitro affinity-isolation assay, we show that SQSTM1 only binds to monomeric MAPT when BAG3 is absent and fails to bind when BAG3 is present. The opposite is true of NBR1; its association with monomeric MAPT was dependent on the presence of BAG3. Interestingly, in Alzheimer disease brain the association of NBR1 with BAG3 was significantly decreased. In a mouse model, ablation of BAG3 in neural cells disrupted the association of NBR1 with phosphorylated MAPT and led to increased levels of phosphorylated and oligomeric MAPT. Overall, our results uncover a novel role for BAG3 in regulating the specificity of selective autophagy receptors in targeting different species of MAPT and provide compelling evidence that BAG3 plays a key role in maintaining MAPT proteostasis.

Keywords:  Alzheimer’s disease; Autophagy receptor; BAG3; MAPT; NBR1; SQSTM1

DOI:  https://doi.org/10.1080/15548627.2023.2276622
Autophagy. 2023 Nov 01.

Control of ATG14 solubility and autophagy by MARCHF7/MARCH7-mediated ubiquitination.

Xue Shi, Xiaofei Zhang.

  Emerging research has unequivocally demonstrated the significance of post-translational modifications (PTMs) of proteins in orchestrating macroautophagy/autophagy regulation. Ubiquitination is a common PTM of proteins that regulates their stability, activity, and localization, thus playing a crucial role in various cellular processes, including autophagy. In recent work, a ubiquitination-related study revealed that MARCHF7/MARCH7 promotes the mixed polyubiquitination of ATG14 at multiple sites, mainly through the linkages of K6, K11, and K63 ubiquitin chains. Consequently, mixed ubiquitination leads to substantial insoluble aggregation of ATG14/ATG14L/Barkor, reducing its interaction with STX17, and ultimately causing a decrease in autophagy flux. It is noteworthy that we have observed that this regulation may hold significant potential value for the autophagic degradation of protein aggregates, as the number of aggresome-like induced structures (ALISs) is markedly reduced in MARCHF7 knockout cells. This may have important potential implications for neurodegenerative diseases characterized by protein aggregation and impaired degradation.

Keywords:  ATG14; Aggregation; MARCHF7; autophagy; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2023.2278414
bioRxiv. 2023 Oct 20. pii: 2023.10.19.563107. [Epub ahead of print]

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

Naoto Soya, Haijin Xu, Ariel Roldan, Zhengrong Yang, Haoxin Ye, Fan Jiang, Aiswarya Premchandar, Guido Veit, Susan P C Cole, John Kappes, Tamas Hegedus, Gergely L Lukacs.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding.One-Sentence Summary: Allosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.

DOI: https://doi.org/10.1101/2023.10.19.563107
Autophagy. 2023 Nov 01. 1-17

SMURF1 controls the PPP3/calcineurin complex and TFEB at a regulatory node for lysosomal biogenesis.

Qin Xia, Hanfei Zheng, Yang Li, Wanting Xu, Chengwei Wu, Jiachen Xu, Shanhu Li, Lingqiang Zhang, Lei Dong.

  Macroautophagy/autophagy is a homeostatic process in response to multiple signaling, such as the lysosome-dependent recycling process of cellular components. Starvation-induced MTOR inactivation and PPP3/calcineurin activation were shown to promote the nuclear translocation of TFEB. However, the mechanisms via which signals from endomembrane damage are transmitted to activate PPP3/calcineurin and orchestrate autophagic responses remain unknown. This study aimed to show that autophagy regulator SMURF1 controlled TFEB nuclear import for transcriptional activation of the lysosomal biogenesis. We showed that blocking SMURF1 affected lysosomal biogenesis in response to lysosomal damage by preventing TFEB nuclear translocation. It revealed galectins recognized endolysosomal damage, and led to recruitment of SMURF1 and the PPP3/calcineurin apparatus on lysosomes. SMURF1 interacts with both LGALS3 and PPP3CB to form the LGALS3-SMURF1-PPP3/calcineurin complex. Importantly, this complex further stabilizes TFEB, thereby activating TFEB for lysosomal biogenesis. We determined that LLOMe-mediated TFEB nuclear import is dependent on SMURF1 under the condition of MTORC1 inhibition. In addition, SMURF1 is required for PPP3/calcineurin activity as a positive regulator of TFEB. SMURF1 controlled the phosphatase activity of the PPP3CB by promoting the dissociation of its autoinhibitory domain (AID) from its catalytic domain (CD). Overexpression of SMURF1 showed similar effects as the constitutive activation of PPP3CB. Thus, SMURF1, which bridges environmental stress with the core autophagosomal and autolysosomal machinery, interacted with endomembrane sensor LGALS3 and phosphatase PPP3CB to control TFEB activation.Abbreviations: ATG: autophagy-related; LLOMe: L-Leucyl-L-Leucine methyl ester; ML-SA1: mucolipin synthetic agonist 1; MTOR: mechanistic target of rapamycin kinase; PPP3CB: protein phosphatase 3 catalytic subunit beta; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; TFEB: transcription factor EB.

Keywords:  Autophagy; PPP3/Calcineurin; SMURF1; TFEB; lysosomal biogenesis

DOI:  https://doi.org/10.1080/15548627.2023.2267413
Nat Chem Biol. 2023 Nov 02.

DNA-encoded library-enabled discovery of proximity-inducing small molecules.

Jeremy W Mason, Yuen Ting Chow, Liam Hudson, Antonin Tutter, Gregory Michaud, Matthias V Westphal, Wei Shu, Xiaolei Ma, Zher Yin Tan, Connor W Coley, Paul A Clemons, Simone Bonazzi, Frédéric Berst, Karin Briner, Shuang Liu, Frédéric J Zécri, Stuart L Schreiber.

Small molecules that induce protein-protein associations represent powerful tools to modulate cell circuitry. We sought to develop a platform for the direct discovery of compounds able to induce association of any two preselected proteins, using the E3 ligase von Hippel-Lindau (VHL) and bromodomains as test systems. Leveraging the screening power of DNA-encoded libraries (DELs), we synthesized ~1 million DNA-encoded compounds that possess a VHL-targeting ligand, a variety of connectors and a diversity element generated by split-and-pool combinatorial chemistry. By screening our DEL against bromodomains in the presence and absence of VHL, we could identify VHL-bound molecules that simultaneously bind bromodomains. For highly barcode-enriched library members, ternary complex formation leading to bromodomain degradation was confirmed in cells. Furthermore, a ternary complex crystal structure was obtained for our most enriched library member with BRD4BD1 and a VHL complex. Our work provides a foundation for adapting DEL screening to the discovery of proximity-inducing small molecules.

DOI: https://doi.org/10.1038/s41589-023-01458-4
Nat Commun. 2023 Nov 01. 14(1): 6992

Recognition and reprogramming of E3 ubiquitin ligase surfaces by α-helical peptides.

Olena S Tokareva, Kunhua Li, Tara L Travaline, Ty M Thomson, Jean-Marie Swiecicki, Mahmoud Moussa, Jessica D Ramirez, Sean Litchman, Gregory L Verdine, John H McGee.

Molecules that induce novel interactions between proteins hold great promise for the study of biological systems and the development of therapeutics, but their discovery has been limited by the complexities of rationally designing interactions between three components, and because known binders to each protein are typically required to inform initial designs. Here, we report a general and rapid method for discovering α-helically constrained (Helicon) polypeptides that cooperatively induce the interaction between two target proteins without relying on previously known binders or an intrinsic affinity between the proteins. We show that Helicons are capable of binding every major class of E3 ubiquitin ligases, which are of great biological and therapeutic interest but remain largely intractable to targeting by small molecules. We then describe a phage-based screening method for discovering "trimerizer" Helicons, and apply it to reprogram E3s to cooperatively bind an enzyme (PPIA), a transcription factor (TEAD4), and a transcriptional coactivator (β-catenin).

DOI: https://doi.org/10.1038/s41467-023-42395-z
Mol Biol Cell. 2023 Oct 30. mbcE23020058

Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes.

Itziar Pinilla-Macua, Alexander Sorkin.

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant fashion by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking and signaling.

DOI: https://doi.org/10.1091/mbc.E23-02-0058
ChemMedChem. 2023 Oct 31. e202300530

Kinetic Modeling of PROTAC-Induced Protein Degradation.

Hongtao Zhao, Frank Narjes.

  Kinetics of the PROTAC-induced protein degradation was modelled with the equilibrium approximation, accounting for the protein recovery rate with a time lag. The simulated kinetic curves resemble what is experimentally observed, and the physical formulas of the half-maximal degradation concentration (DC50) were derived from them. The equations reveal that DC50 is proportional to the dissociation constant of the ternary complex (Kd) and inversely proportional to the expression level of the E3 ligase and the effective ubiquitylation rate (kub). The predicted relationships were rigorously confirmed by experimental evidences from a matched molecular pair analysis using a set of published PROTACs.

Keywords:  PROTAC; kinetics; protein degradation; ternary complex; ubiquitylation

DOI:  https://doi.org/10.1002/cmdc.202300530
bioRxiv. 2023 Oct 19. pii: 2023.10.17.562832. [Epub ahead of print]

Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics.

Tianyang Yan, Lisa M Boatner, Liujuan Cui, Peter Tontonoz, Keriann M Backus.

The plasma membrane proteome is a rich resource of functional and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surf ace Cys teinome. Our "Cys-Surf" platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of LDL particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.

DOI: https://doi.org/10.1101/2023.10.17.562832
bioRxiv. 2023 Oct 17. pii: 2023.10.13.562283. [Epub ahead of print]

Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline.

Kelly G Sullivan, Greg J Bashaw.

In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila , Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. Here, we demonstrate that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline in vivo . In addition, we conclusively demonstrate a role for Nedd4 in midline crossing. Genetic analysis shows that nedd4 mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo in a PY motif-dependent manner. Finally, we present genetic evidence that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo ubiquitination by Nedd4, ultimately leading to its degradation.

DOI: https://doi.org/10.1101/2023.10.13.562283
J Biol Chem. 2023 Oct 31. pii: S0021-9258(23)02444-4. [Epub ahead of print] 105416

SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.

Cunjie Zhang, Massiullah Shafaq-Zadah, Judy Pawling, Geoffrey G Hesketh, Estelle Dransart, Karina Pacholczyk, Joseph Longo, Anne-Claude Gingras, Linda Z Penn, Ludger Johannes, James W Dennis.

  Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasing dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism. Herein, we report that each of the four sites in SLC3A2 have distinct profiles of Golgi-modified N-glycans. N-glycans at the primate-derived site N381 stabilized SLC3A2 in the galectin-3 lattice against coated-pit endocytosis, while N365, the site nearest the membrane promoted glycolipid-galectin-3 (GL-Lect) driven endocytosis. Our results indicate that surface retention and endocytosis are precisely balanced by the number, position, and remodeling of N-glycans on SLC3A2. Furthermore, proteomics and functional assays revealed an N-glycan-dependent clustering of the SLC3A2*SLC7A5 heterodimer with amino-acid /Na+ symporters (SLC1A4, SLC1A5) that balances branched-chain amino acids and Gln levels, at the expense of ATP to maintain the Na+/K+ gradient. In replete conditions, SLC3A2 interactions require Golgi-modified N-glycans at N365D and N381D, whereas reducing N-glycosylation in the endoplasmic reticulum by fluvastatin treatment promoted the recruitment of CD44 and transporters needed to mitigate stress. Thus, SLC3A2 N-glycosylation and Golgi remodeling of the N-glycans have distinct roles in amino acids import for growth, maintenance, and metabolic stresses.

Keywords:  Amino acids; evolution; galectins; metabolism; transporters N-glycosylation

DOI:  https://doi.org/10.1016/j.jbc.2023.105416
J Cell Biol. 2023 Dec 04. pii: e202306120. [Epub ahead of print]222(12):

The mechanism of Atg15-mediated membrane disruption in autophagy.

Yoko Kagohashi, Michiko Sasaki, Alexander I May, Tomoko Kawamata, Yoshinori Ohsumi.

Autophagy is a lysosomal/vacuolar delivery system that degrades cytoplasmic material. During autophagy, autophagosomes deliver cellular components to the vacuole, resulting in the release of a cargo-containing autophagic body (AB) into the vacuole. AB membranes must be disrupted for degradation of cargo to occur. The lipase Atg15 and vacuolar proteases Pep4 and Prb1 are known to be necessary for this disruption and cargo degradation, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and show that Atg15 is the sole vacuolar phospholipase. Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments reveal that Atg15 is a phospholipase B of broad substrate specificity that is likely implicated in the disruption of a range of membranes. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.

DOI: https://doi.org/10.1083/jcb.202306120
Biochimie. 2023 Oct 31. pii: S0300-9084(23)00290-0. [Epub ahead of print]

Crosstalk between protein misfolding and endoplasmic reticulum stress during ageing and their role in age-related disorders.

Manisekaran Hemagirri, Yeng Chen, Subash C B Gopinath, Sumaira Sahreen, Mohd Adnan, Sreenivasan Sasidharan.

  Maintaining the proteome is crucial to retaining cell functionality and response to multiple intrinsic and extrinsic stressors. Protein misfolding increased the endoplasmic reticulum (ER) stress and activated the adaptive unfolded protein response (UPR) to restore cell homeostasis. Apoptosis occurs when ER stress is prolonged or the adaptive response fails. In healthy young cells, the ratio of protein folding machinery to quantities of misfolded proteins is balanced under normal circumstances. However, the age-related deterioration of the complex systems for handling protein misfolding is accompanied by ageing-related disruption of protein homeostasis, which results in the build-up of misfolded and aggregated proteins. This ultimately results in decreased cell viability and forms the basis of common age-related diseases called protein misfolding diseases. Proteins or protein fragments convert from their ordinarily soluble forms to insoluble fibrils or plaques in many of these disorders, which build up in various organs such as the liver, brain, or spleen. Alzheimer's, Parkinson's, type II diabetes, and cancer are diseases in this group commonly manifest in later life. Thus, protein misfolding and its prevention by chaperones and different degradation paths are becoming understood from molecular perspectives. Proteodynamics information will likely affect future interventional techniques to combat cellular stress and support healthy ageing by avoiding and treating protein conformational disorders. This review provides an overview of the diverse proteostasis machinery, protein misfolding, and ER stress involvement, which activates the UPR sensors. Here, we will discuss the crosstalk between protein misfolding and ER stress and their role in developing age-related diseases.

Keywords:  Age-related diseases; Ageing; Endoplasmic reticulum stress response; Protein misfolding; Proteostasis; Unfolded protein response

DOI:  https://doi.org/10.1016/j.biochi.2023.10.019
J Clin Invest. 2023 Nov 02. pii: e168544. [Epub ahead of print]

CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses.

Josephine W Thinwa, Zhongju Zou, Emily Parks, Salwa Sebti, Kelvin K Hui, Yongjie Wei, Mohammad Goodarzi, Vibha Singh, Greg Urquhart, Jenna L Jewell, Julie K Pfeiffer, Beth Levine, Tiffany A Reese, Michael U Shiloh.

  Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase-like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss of function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found deletion of CDKL5 or expression of a clinically-relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. CDKL5 knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.

Keywords:  Autophagy; Cellular immune response; Infectious disease; Innate immunity; Virology

DOI:  https://doi.org/10.1172/JCI168544
Nat Cancer. 2023 Oct 30.

Structural surfaceomics reveals an AML-specific conformation of integrin β2 as a CAR T cellular therapy target.

Kamal Mandal, Gianina Wicaksono, Clinton Yu, Jarrett J Adams, Michael R Hoopmann, William C Temple, Adila Izgutdina, Bonell Patiño Escobar, Maryna Gorelik, Christian H Ihling, Matthew A Nix, Akul Naik, William H Xie, Juwita Hübner, Lisa A Rollins, Sandy M Reid, Emilio Ramos, Corynn Kasap, Veronica Steri, Juan Antonio Camara Serrano, Fernando Salangsang, Paul Phojanakong, Melanie McMillan, Victor Gavallos, Andrew D Leavitt, Aaron C Logan, Cliona M Rooney, Justin Eyquem, Andrea Sinz, Benjamin J Huang, Elliot Stieglitz, Catherine C Smith, Robert L Moritz, Sachdev S Sidhu, Lan Huang, Arun P Wiita.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin β2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

DOI: https://doi.org/10.1038/s43018-023-00652-6
Nat Commun. 2023 Oct 31. 14(1): 6931

Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells.

Mikhail E Sushkin, Christine Koehler, Edward A Lemke.

Genetic code expansion (GCE) reprograms the translational machinery to site-specifically incorporate noncanonical amino acids (ncAAs) into a selected protein. The efficiency of GCE in mammalian cells might be compromised by cellular stress responses, among which, the protein kinase R(PKR)-dependent eIF2α phosphorylation pathway can reduce translation rates. Here we test several strategies to engineer the eIF2α pathway and boost the rate of translation and show that such interventions increase GCE efficiency in mammalian cells. In particular, addition of the N-terminal PKR fragment (1-174) provides a substantial enhancement in cytoplasmic GCE and also in GCE realized by OTOs (orthogonally translating designer organelles), which built on the principle of 2D phase separation to enable mRNA-selective ncAA incorporation. Our study demonstrates an approach for improving the efficiency of GCE and provides a means by which the power of designer organelles can be further optimized to tune protein translation.

DOI: https://doi.org/10.1038/s41467-023-42689-2
Proc Natl Acad Sci U S A. 2023 Nov 07. 120(45): e2310057120

Shift of the insoluble content of the proteome in the aging mouse brain.

Cristen Molzahn, Erich R Kuechler, Irina Zemlyankina, Lorenz Nierves, Tahir Ali, Grace Cole, Jing Wang, Razvan F Albu, Mang Zhu, Neil R Cashman, Sabine Gilch, Aly Karsan, Philipp F Lange, Jörg Gsponer, Thibault Mayor.

  During aging, the cellular response to unfolded proteins is believed to decline, resulting in diminished proteostasis. In model organisms, such as Caenorhabditis elegans, proteostatic decline with age has been linked to proteome solubility shifts and the onset of protein aggregation. However, this correlation has not been extensively characterized in aging mammals. To uncover age-dependent changes in the insoluble portion of a mammalian proteome, we analyzed the detergent-insoluble fraction of mouse brain tissue by mass spectrometry. We identified a group of 171 proteins, including the small heat shock protein α-crystallin, that become enriched in the detergent-insoluble fraction obtained from old mice. To enhance our ability to detect features associated with proteins in that fraction, we complemented our data with a meta-analysis of studies reporting the detergent-insoluble proteins in various mouse models of aging and neurodegeneration. Strikingly, insoluble proteins from young and old mice are distinct in several features in our study and across the collected literature data. In younger mice, proteins are more likely to be disordered, part of membraneless organelles, and involved in RNA binding. These traits become less prominent with age, as an increased number of structured proteins enter the pellet fraction. This analysis suggests that age-related changes to proteome organization lead a group of proteins with specific features to become detergent-insoluble. Importantly, these features are not consistent with those associated with proteins driving membraneless organelle formation. We see no evidence in our system of a general increase of condensate proteins in the detergent-insoluble fraction with age.

Keywords:  aggregation; aging; neurodegeneration; protein homeostasis; proteomics

DOI:  https://doi.org/10.1073/pnas.2310057120
J Mol Biol. 2023 Oct 31. pii: S0022-2836(23)00448-5. [Epub ahead of print] 168337

What strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.

Ta I Hung, Yun-Jung Hsieh, Wei-Lin Lu, Kuen-Phon Wu, Chia-En A Chang.

  Identifying residues critical to protein-protein binding and efficient design of stable and specific protein binders are challenging tasks. Extending beyond the direct contacts in a protein-protein binding interface, our study employs computational modeling to reveal the essential network of residue interactions and dihedral angle correlations critical in protein-protein recognition. We hypothesized that mutating residues exhibiting highly correlated dynamic motion within the interaction network could efficiently optimize protein-protein interactions to create tight and selective protein binders. We tested this hypothesis using the ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complex, since Ub is a central player in multiple cellular functions and PLpro is an antiviral drug target. Our designed ubiquitin variant (UbV) hosting three mutated residues displayed a ∼3,500-fold increase in functional inhibition relative to wild-type Ub. Further optimization of two C-terminal residues within the Ub network resulted in a KD of 1.5 nM and IC50 of 9.7 nM for the five-point Ub mutant, eliciting 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study highlights residue correlation and interaction networks in protein-protein interactions, and introduces an effective approach to design high-affinity protein binders for cell biology research and future therapeutics.

Keywords:  Dihedral angle correlation network; nanomolar binder; papain-like protease; protein-based inhibitors; ubiquitin variant

DOI:  https://doi.org/10.1016/j.jmb.2023.168337
PLoS Genet. 2023 Oct 31. 19(10): e1011014

Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs.

Tiit Örd, Daima Örd, Priit Adler, Tõnis Örd.

Activating Transcription Factor 4 (ATF4) is an important regulator of gene expression in stress responses and developmental processes in many cell types. Here, we catalogued ATF4 binding sites in the human genome and identified overlaps with trait-associated genetic variants. We probed these genetic variants for allelic regulatory activity using a massively parallel reporter assay (MPRA) in HepG2 hepatoma cells exposed to tunicamycin to induce endoplasmic reticulum stress and ATF4 upregulation. The results revealed that in the majority of cases, the MPRA allelic activity of these SNPs was in agreement with the nucleotide preference seen in the ATF4 binding motif from ChIP-Seq. Luciferase and electrophoretic mobility shift assays in additional cellular models further confirmed ATF4-dependent regulatory effects for the SNPs rs532446 (GADD45A intronic; linked to hematological parameters), rs7011846 (LPL upstream; myocardial infarction), rs2718215 (diastolic blood pressure), rs281758 (psychiatric disorders) and rs6491544 (educational attainment). CRISPR-Cas9 disruption and/or deletion of the regulatory elements harboring rs532446 and rs7011846 led to the downregulation of GADD45A and LPL, respectively. Thus, these SNPs could represent examples of GWAS genetic variants that affect gene expression by altering ATF4-mediated transcriptional activation.

DOI: https://doi.org/10.1371/journal.pgen.1011014