bims-proteo 2023-02-26 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒02‒26
34 papers selected by
Eric Chevet
INSERM

Macro-ER-phagy receptors Atg39p and Atg40p confer resistance to aminoglycoside hygromycin B in S. cerevisiae.
Molecular basis of eIF5A-dependent CAT tailing in eukaryotic ribosome-associated quality control.
TRIM21-dependent target protein ubiquitination mediates cell-free Trim-Away.
Co-Translational Quality Control Induced by Translational Arrest.
Ubiquitin and its relatives as wizards of the endolysosomal system.
Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae.
Targeting of client proteins to the VCP/p97/Cdc48 unfolding machine.
Fitm2 is required for ER homeostasis and normal function of murine liver.
Emerging roles of the HECT E3 ubiquitin ligases in gastric cancer.
E3 Ligases Meet Their Match: Fragment-Based Approaches to Discover New E3 Ligands and to Unravel E3 Biology.
A deep dive into degrader-induced protein-protein interfaces.
Structural basis for clearing of ribosome collisions by the RQT complex.
Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.
E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity.
Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.
Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.
E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase.
The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells.
Protein disulfide isomerases as CSF biomarkers for the neuronal response to tau pathology.
The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction.
Post-ovulatory aging is associated with altered patterns for small ubiquitin-like modifier (SUMO) proteins and SUMO-specific proteases.
Human gene-engineered calreticulin mutant stem cells recapitulate MPN hallmarks and identify targetable vulnerabilities.
Extracellular protein homeostasis in neurodegenerative diseases.
Exploiting the intrinsic misfolding propensity of the KRAS oncoprotein.
Proteasomal pathway inhibition as a potential therapy for NF2-associated meningioma and schwannoma.
Multiple ubiquitin E3 ligase genes antagonistically regulate chloroplast-associated protein degradation.
Breaking free from the crystal lattice: Structural biology in solution to study protein degraders.
Spatial position is a key determinant of N-glycan functionality of the scavenger receptor cysteine-rich domain of human hepsin.
Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria.
The p53 endoplasmic reticulum stress-response pathway evolved in humans but not in mice via PERK-regulated p53 mRNA structures.
Identification of global inhibitors of cellular glycosylation.
Protein complexes in cells by AI-assisted structural proteomics.
mRNA interactions with disordered regions control protein activity.
Periostin protects against alcohol-related liver disease by activating autophagy by interacting with PDI.

MicroPubl Biol. 2023 ;2023

Macro-ER-phagy receptors Atg39p and Atg40p confer resistance to aminoglycoside hygromycin B in S. cerevisiae.

Mahmoud M Daraghmi, Jacob M Miller, Connor G Bailey, Ellen M Doss, Ashley L Kalinski, Philip J Smaldino, Eric M Rubenstein.

Receptor-mediated autophagic turnover of portions of the endoplasmic reticulum (ER) is mediated by macro-ER-phagy. We hypothesized macro-ER-phagy promotes proteotoxic stress resistance. We predicted Saccharomyces cerevisiae lacking macro-ER-phagy receptors would exhibit enhanced sensitivity to hygromycin B, which reduces translational fidelity and is expected to globally disrupt protein homeostasis, including at the ER. We observed that loss of either of two yeast macro-ER-phagy receptors (Atg39p or Atg40p) compromised cellular resistance to hygromycin B to a similar extent as loss of ER-associated degradation (ERAD) ubiquitin ligases Hrd1p and Doa10p. Our data are consistent with a model whereby macro-ER-phagy and ERAD collaborate to mediate ER protein quality control. Disruptions of macro-ER-phagy have been linked to neuropathy, dementia, and cancer. A dampened capacity to mediate protein quality control may contribute to these conditions.

DOI: https://doi.org/10.17912/micropub.biology.000738
Mol Cell. 2023 Feb 16. pii: S1097-2765(23)00072-2. [Epub ahead of print]83(4): 607-621.e4

Molecular basis of eIF5A-dependent CAT tailing in eukaryotic ribosome-associated quality control.

Petr Tesina, Shuhei Ebine, Robert Buschauer, Matthias Thoms, Yoshitaka Matsuo, Toshifumi Inada, Roland Beckmann.

  Ribosome-associated quality control (RQC) is a conserved process degrading potentially toxic truncated nascent peptides whose malfunction underlies neurodegeneration and proteostasis decline in aging. During RQC, dissociation of stalled ribosomes is followed by elongation of the nascent peptide with alanine and threonine residues, driven by Rqc2 independently of mRNA, the small ribosomal subunit and guanosine triphosphate (GTP)-hydrolyzing factors. The resulting CAT tails (carboxy-terminal tails) and ubiquitination by Ltn1 mark nascent peptides for proteasomal degradation. Here we present ten cryogenic electron microscopy (cryo-EM) structures, revealing the mechanistic basis of individual steps of the CAT tailing cycle covering initiation, decoding, peptidyl transfer, and tRNA translocation. We discovered eIF5A as a crucial eukaryotic RQC factor enabling peptidyl transfer. Moreover, we observed dynamic behavior of RQC factors and tRNAs allowing for processivity of the CAT tailing cycle without additional energy input. Together, these results elucidate key differences as well as common principles between CAT tailing and canonical translation.

Keywords:  Listerin; Ltn1; NEMF; Rqc1; Rqc2; eIF5A; peptidyl transfer, P/P(∗) tRNA; ribosome-associated quality control; translation

DOI:  https://doi.org/10.1016/j.molcel.2023.01.020
Cell Rep. 2023 Feb 18. pii: S2211-1247(23)00136-5. [Epub ahead of print]42(2): 112125

TRIM21-dependent target protein ubiquitination mediates cell-free Trim-Away.

Tycho E T Mevissen, Anisa V Prasad, Johannes C Walter.

  Tripartite motif-containing protein 21 (TRIM21) is a cytosolic antibody receptor and E3 ubiquitin ligase that promotes destruction of a broad range of pathogens. TRIM21 also underlies the antibody-dependent protein targeting method Trim-Away. Current evidence suggests that TRIM21 binding to antibodies leads to formation of a self-anchored K63 ubiquitin chain on the N terminus of TRIM21 that triggers the destruction of TRIM21, antibody, and target protein. Here, we report that addition of antibody and TRIM21 to Xenopus egg extracts promotes efficient degradation of endogenous target proteins, establishing cell-free Trim-Away as a powerful tool to interrogate protein function. Chemical methylation of TRIM21 had no effect on target proteolysis, whereas deletion of all lysine residues in targets abolished their ubiquitination and proteasomal degradation. These results demonstrate that target protein, but not TRIM21, polyubiquitination is required for Trim-Away, and they suggest that current models of TRIM21 function should be fundamentally revised.

Keywords:  CP: Molecular biology; TRIM21; Trim-Away; Ubiquitin; Xenopus egg extract; targeted protein degradation

DOI:  https://doi.org/10.1016/j.celrep.2023.112125
Biomolecules. 2023 Feb 07. pii: 317. [Epub ahead of print]13(2):

Co-Translational Quality Control Induced by Translational Arrest.

Yoshitaka Matsuo, Toshifumi Inada.

  Genetic mutations, mRNA processing errors, and lack of availability of charged tRNAs sometimes slow down or completely stall translating ribosomes. Since an incomplete nascent chain derived from stalled ribosomes may function anomalously, such as by forming toxic aggregates, surveillance systems monitor every step of translation and dispose of such products to prevent their accumulation. Over the past decade, yeast models with powerful genetics and biochemical techniques have contributed to uncovering the mechanism of the co-translational quality control system, which eliminates the harmful products generated from aberrant translation. We here summarize the current knowledge of the molecular mechanism of the co-translational quality control systems in yeast, which eliminate the incomplete nascent chain, improper mRNAs, and faulty ribosomes to maintain cellular protein homeostasis.

Keywords:  mRNA decay; non-canonical ribosome dissociation; protein degradation; quality control; ribosome collision; transrational arrest; ubiquitination

DOI:  https://doi.org/10.3390/biom13020317
J Cell Sci. 2023 Feb 15. pii: jcs260101. [Epub ahead of print]136(4):

Ubiquitin and its relatives as wizards of the endolysosomal system.

Ilana Berlin, Aysegul Sapmaz, Virginie Stévenin, Jacques Neefjes.

  The endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information with the internal affairs of the cell requires endosomes and lysosomes to be proficient in decision-making: fusion or fission; recycling or degradation; fast transport or contacts with other organelles. To effectively discriminate between these options, the endolysosomal system employs complex regulatory strategies that crucially rely on reversible post-translational modifications (PTMs) with ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. The cycle of conjugation, recognition and removal of different Ub- and Ubl-modified states informs cellular protein stability and behavior at spatial and temporal resolution and is thus well suited to finetune macromolecular complex assembly and function on endolysosomal membranes. Here, we discuss how ubiquitylation (also known as ubiquitination) and its biochemical relatives orchestrate endocytic traffic and designate cargo fate, influence membrane identity transitions and support formation of membrane contact sites (MCSs). Finally, we explore the opportunistic hijacking of Ub and Ubl modification cascades by intracellular bacteria that remodel host trafficking pathways to invade and prosper inside cells.

Keywords:  Bacterial infection; Endosomes; Membrane contact sites; Membrane dynamics; Ubiquitin

DOI:  https://doi.org/10.1242/jcs.260101
Autophagy. 2023 Feb 20.

Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae.

Yongook Lee, Bongkeun Kim, Hae-Soo Jang, Won-Ki Huh.

  Macroautophagy/autophagy is a key catabolic pathway in which double-membrane autophagosomes sequester various substrates destined for degradation, enabling cells to maintain homeostasis and survive under stressful conditions. Several autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS) and cooperatively function to generate autophagosomes. Vps34 is a class III phosphatidylinositol 3-kinase, and Atg14-containing Vps34 complex I plays essential roles in autophagosome formation. However, the regulatory mechanisms of yeast Vps34 complex I are still poorly understood. Here, we demonstrate that Atg1-dependent phosphorylation of Vps34 is required for robust autophagy activity in Saccharomyces cerevisiae. Following nitrogen starvation, Vps34 in complex I is selectively phosphorylated on multiple serine/threonine residues in its helical domain. This phosphorylation is important for full autophagy activation and cell survival. The absence of Atg1 or its kinase activity leads to complete loss of Vps34 phosphorylation in vivo, and Atg1 directly phosphorylates Vps34 in vitro, regardless of its complex association type. We also demonstrate that the localization of Vps34 complex I to the PAS provides a molecular basis for the complex I-specific phosphorylation of Vps34. This phosphorylation is required for the normal dynamics of Atg18 and Atg8 at the PAS. Together, our results reveal a novel regulatory mechanism of yeast Vps34 complex I and provide new insights into the Atg1-dependent dynamic regulation of the PAS.

Keywords:  Atg1; Atg18; Atg8; Saccharomyces cerevisiae; Vps34; autophagy; nitrogen starvation

DOI:  https://doi.org/10.1080/15548627.2023.2182478
Front Mol Biosci. 2023 ;10 1142989

Targeting of client proteins to the VCP/p97/Cdc48 unfolding machine.

Hemmo Meyer, Johannes van den Boom.

  The AAA+ ATPase p97 (also called VCP or Cdc48) is a major protein unfolding machine with hundreds of clients in diverse cellular pathways that are critical for cell homeostasis, proliferation and signaling. In this review, we summarize recent advances in understanding how diverse client proteins are targeted to the p97 machine to facilitate client degradation or to strip clients from binding partners for regulation. We describe an elaborate system that is governed by at least two types of alternative adapters. The Ufd1-Npl4 adapter along with accessory adapters targets ubiquitylated clients in the majority of pathways and uses ubiquitin as a universal unfolding tag. In contrast, the family of SEP-domain adapters such as p37 can target clients directly to p97 in a ubiquitin-independent manner. Despite the different targeting strategies, both pathways converge by inserting the client into the p97 pore to initiate a peptide threading mechanism through the central channel of p97 that drives client protein unfolding, protein extraction from membranes and protein complex disassembly processes.

Keywords:  DNA replication and damaged repair; protein homeostasis; protein phosphatase (PP) 1; protein quality control; protein unfolding; ubiquitin

DOI:  https://doi.org/10.3389/fmolb.2023.1142989
J Biol Chem. 2023 Feb 15. pii: S0021-9258(23)00154-0. [Epub ahead of print] 103022

Fitm2 is required for ER homeostasis and normal function of murine liver.

Laura M Bond, Ayon Ibrahim, Zon W Lai, Rosemary L Walzem, Roderick T Bronson, Olga R Ilkayeva, Tobias C Walther, Robert V Farese.

  The endoplasmic reticulum (ER)-resident protein fat storage-inducing transmembrane protein 2 (FIT2) catalyzes acyl-CoA cleavage in vitro and is required for ER homeostasis and normal lipid storage in cells. The gene encoding FIT2 is essential for the viability of mice and worms. Whether FIT2 acts as an acyl-CoA diphosphatase in vivo and how this activity affects the liver, where the protein was discovered, are unknown. Here, we report that hepatocyte-specific Fitm2 knockout (FIT2-LKO) mice fed a chow diet exhibited elevated acyl-CoA levels, ER stress, and signs of liver injury. These mice also had more triglycerides in their livers than control littermates due, in part, to impaired secretion of triglyceride-rich lipoproteins and reduced capacity for fatty acid oxidation. We found that challenging FIT2-LKO mice with a high-fat diet worsened hepatic ER stress and liver injury, but unexpectedly reversed the steatosis phenotype, similar to what is observed in FIT2-deficient cells loaded with fatty acids. Our findings support the model that FIT2 acts as an acyl-CoA diphosphatase in vivo and is crucial for normal hepatocyte function and ER homeostasis in murine liver.

Keywords:  FITM2; acyl-CoA; endoplasmic reticulum; lipid metabolism; liver

DOI:  https://doi.org/10.1016/j.jbc.2023.103022
Pathol Oncol Res. 2023 ;29 1610931

Emerging roles of the HECT E3 ubiquitin ligases in gastric cancer.

Aiqin Sun, Xianyan Tian, Yifei Chen, Wannian Yang, Qiong Lin.

  Gastric cancer (GC) is one of the most pernicious gastrointestinal tumors with extraordinarily high incidence and mortality. Ubiquitination modification of cellular signaling proteins has been shown to play important roles in GC tumorigenesis, progression, and prognosis. The E3 ubiquitin ligase is the crucial enzyme in the ubiquitination reaction and determines the specificity of ubiquitination substrates, and thus, the cellular effects. The HECT E3 ligases are the second largest E3 ubiquitin ligase family characterized by containing a HECT domain that has E3 ubiquitin ligase activity. The HECT E3 ubiquitin ligases have been found to engage in GC progression. However, whether HECT E3 ligases function as tumor promoters or tumor suppressors in GC remains controversial. In this review, we will focus on recent discoveries about the role of the HECT E3 ubiquitin ligases, especially members of the NEDD4 and other HECT E3 ligase subfamilies, in GC.

Keywords:  HECT E3 ubiquitin ligases; gastric cancer; oncoprotein; tumor suppressor; ubiquitination

DOI:  https://doi.org/10.3389/pore.2023.1610931
J Med Chem. 2023 Feb 23.

E3 Ligases Meet Their Match: Fragment-Based Approaches to Discover New E3 Ligands and to Unravel E3 Biology.

Iacovos N Michaelides, Gavin W Collie.

Ubiquitination is a key post-translational modification of proteins, affecting the regulation of multiple cellular processes. Cells are equipped with over 600 ubiquitin orchestrators, called E3 ubiquitin ligases, responsible for directing the covalent attachment of ubiquitin to substrate proteins. Due to their regulatory role in cells, significant efforts have been made to discover ligands for E3 ligases. The recent emergence of the proteolysis targeting chimera (PROTAC) and molecular glue degrader (MGD) modalities has further increased interest in E3 ligases as drug targets. This perspective focuses on how fragment based lead discovery (FBLD) methods have been used to discover new ligands for this important target class. In some cases these efforts have led to clinical candidates; in others, they have provided tools for deepening our understanding of E3 ligase biology. Recently, FBLD-derived ligands have inspired the design of PROTACs that are able to artificially modulate protein levels in cells.

DOI: https://doi.org/10.1021/acs.jmedchem.2c01882
Trends Pharmacol Sci. 2023 Feb 22. pii: S0165-6147(23)00035-4. [Epub ahead of print]

A deep dive into degrader-induced protein-protein interfaces.

Pius Galli, Carlos Pla-Prats, Nicolas H Thomä.

  Targeted protein degradation (TPD) relies on a comprehensive understanding of interfaces between hijacked E3 ligases and their substrates. In vitro techniques often do not capture the interaction dynamics. Recently, Hanzl et al. introduced deep mutational scanning (DMS) in combination with structural and biochemical approaches to identify residues crucial for degrader activity.

Keywords:  degrader resistance; saturation mutagenesis; targeted protein degradation; ubiquitin ligase

DOI:  https://doi.org/10.1016/j.tips.2023.02.001
Nat Commun. 2023 Feb 17. 14(1): 921

Structural basis for clearing of ribosome collisions by the RQT complex.

Katharina Best, Ken Ikeuchi, Lukas Kater, Daniel Best, Joanna Musial, Yoshitaka Matsuo, Otto Berninghausen, Thomas Becker, Toshifumi Inada, Roland Beckmann.

Translation of aberrant messenger RNAs can cause stalling of ribosomes resulting in ribosomal collisions. Collided ribosomes are specifically recognized to initiate stress responses and quality control pathways. Ribosome-associated quality control facilitates the degradation of incomplete translation products and requires dissociation of the stalled ribosomes. A central event is therefore the splitting of collided ribosomes by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryogenic electron microscopy of RQT-ribosome complexes reveals that RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose that the Ski2-like helicase 1 (Slh1) subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the small ribosomal subunit, ultimately resulting in subunit dissociation. Our findings provide conceptual framework for a helicase-driven ribosomal splitting mechanism.

DOI: https://doi.org/10.1038/s41467-023-36230-8
J Biol Chem. 2023 Feb 21. pii: S0021-9258(23)00189-8. [Epub ahead of print] 103057

Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.

MorganA Gingerich, Jie Zhu, Biaoxin Chai, MichaelP Vincent, Nuli Xie, Vaibhav Sidarala, NicholasA Kotov, Debashish Sahu, DanielJ Klionsky, Santiago Schnell, ScottA Soleimanpour.

CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover. IDPRs lack a fixed secondary structure and possess emerging, yet still equivocal roles in protein stability, interactions, and enzymatic activity. We find that the internal IDPR of CLEC16A is crucial for its degradation. CLEC16A turnover was promoted by RNF41, which binds and acts upon the internal IDPR to destabilize CLEC16A. Loss of this internal IDPR also destabilized the ubiquitin-dependent tripartite CLEC16A-RNF41-USP8 complex. Finally, the presence of an internal IDPR within CLEC16A was confirmed using NMR and circular dichroism spectroscopy. Together, our studies reveal that an IDPR is essential to control the reciprocal regulatory balance between CLEC16A and RNF41, which could be targeted to improve mitochondrial health in disease.

DOI: https://doi.org/10.1016/j.jbc.2023.103057
Mol Cell. 2023 Feb 10. pii: S1097-2765(23)00042-4. [Epub ahead of print]

E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity.

Daniel C Scott, Moeko T King, Kheewoong Baek, Clifford T Gee, Ravi Kalathur, Jerry Li, Nicholas Purser, Amanda Nourse, Sergio C Chai, Sivaraja Vaithiyalingam, Taosheng Chen, Richard E Lee, Stephen J Elledge, Gary Kleiger, Brenda A Schulman.

  E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

Keywords:  Autoinhibition; C-END degron; CUL2; Cullin-RING Ligase; E3; KLHDC10; KLHDC3; Kinetic proofreading; NEDD8; Targeted protein degradation; allostery; higher-order assembly; protein-protein interaction; ubiquitin

DOI:  https://doi.org/10.1016/j.molcel.2023.01.019
J Cell Biol. 2023 May 01. pii: e202208159. [Epub ahead of print]222(5):

Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.

Yi-Shi Liu, Yicheng Wang, Xiaoman Zhou, Linpei Zhang, Ganglong Yang, Xiao-Dong Gao, Yoshiko Murakami, Morihisa Fujita, Taroh Kinoshita.

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.

DOI: https://doi.org/10.1083/jcb.202208159
bioRxiv. 2023 Feb 16. pii: 2023.02.15.528768. [Epub ahead of print]

Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.

Yuanhui Mao, Longfei Jia, Leiming Dong, Xin Erica Shu, Shu-Bing Qian.

A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation immediately from the start codon. The start codon-associated ribosome frameshifting (SCARF) stems from the slippage of ribosomes during the transition from initiation to elongation. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra unannotated from human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity by stabilizing initiating ribosomes. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF protects cells from starvation by enabling amino acid recycling and selective mRNA translation. Our findings illustrate a beneficial effect of translational "noise" in nutrient stress adaptation.

DOI: https://doi.org/10.1101/2023.02.15.528768
Cancers (Basel). 2023 Feb 07. pii: 1040. [Epub ahead of print]15(4):

E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase.

Georgia N Delegkou, Maria Birkou, Nefeli Fragkaki, Tamara Toro, Konstantinos D Marousis, Vasso Episkopou, Georgios A Spyroulias.

  Arkadia (RNF111) is a positive regulator of the TGF-β signaling that mediates the proteasome-dependent degradation of negative factors of the pathway. It is classified as an E3 ubiquitin ligase and a SUMO-targeted ubiquitin ligase (STUBL), implicated in various pathological conditions including cancer and fibrosis. The enzymatic (ligase) activity of Arkadia is located at its C-terminus and involves the RING domain. Notably, E3 ligases require E2 enzymes to perform ubiquitylation. However, little is known about the cooperation of Arkadia with various E2 enzymes and the type of ubiquitylation that they mediate. In the present work, we study the interaction of Arkadia with the E2 partners UbcH5B and UbcH13, as well as UbcH7. Through NMR spectroscopy, we found that the E2-Arkadia interaction surface is similar in all pairs examined. Nonetheless, the requirements and factors that determine an enzymatically active E2-Arkadia complex differ in each case. Furthermore, we revealed that the cooperation of Arkadia with different E2s results in either monoubiquitylation or polyubiquitin chain formation via K63, K48, or K11 linkages, which can determine the fate of the substrate and lead to distinct biological outcomes.

Keywords:  Arkadia; E2 enzymes; NMR spectroscopy; RING domain; ubiquitylation

DOI:  https://doi.org/10.3390/cancers15041040
Autophagy. 2023 Feb 22. 1-21

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells.

Elif Begüm Gökerküçük, Angélique Cheron, Marc Tramier, Giulia Bertolin.

  Although several mechanisms of macroautophagy/autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to its activation, the ATG4B protease primes the key autophagy player MAP1LC3B/LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster's resonance energy transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine-tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image makes it possible to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are pools of unprimed LC3B upon ATG4B downregulation, and the priming of the biosensor is abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Moreover, we screened for commercially-available ATG4B inhibitors, and illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Finally, we uncovered the CDK1-dependent regulation of the ATG4B-LC3B axis at mitosis. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.Abbreviations: Aqua: aquamarine; ATG: autophagy related; AURKA: aurora kinase A; BafA1: bafilomycin A1; CDK1: cyclin dependent kinase 1; DKO: double knockout; FLIM: fluorescence lifetime imaging microscopy; FP: fluorescence protein; FRET: Förster's resonance energy transfer; GABARAP: GABA type A receptor-associated protein; HBSS: Hanks' balanced salt solution; KO: knockout; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSC: NSC 185058; PE: phosphatidylethanolamine; SKO: single knockout; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type; ZPCK: Z-L-phe chloromethyl ketone.

Keywords:  ATG4B; FRET-FLIM; LC3B; autophagy; biosensor

DOI:  https://doi.org/10.1080/15548627.2023.2179845
Alzheimers Dement. 2023 Feb 24.

Protein disulfide isomerases as CSF biomarkers for the neuronal response to tau pathology.

Kimberly Wolzak, Lisa Vermunt, Marta Del Campo, Marta Jorge-Oliva, Anna Maria van Ziel, Ka Wan Li, August B Smit, Alice Chen-Ploktkin, David J Irwin, Afina W Lemstra, Yolande Pijnenburg, Wiesje van der Flier, Henrik Zetterberg, Johan Gobom, Kaj Blennow, Pieter Jelle Visser, Charlotte E Teunissen, Betty M Tijms, Wiep Scheper.

  INTRODUCTION: Cerebrospinal fluid (CSF) biomarkers for specific cellular disease processes are lacking for tauopathies. In this translational study we aimed to identify CSF biomarkers reflecting early tau pathology-associated unfolded protein response (UPR) activation.METHODS: We employed mass spectrometry proteomics and targeted immunoanalysis in a combination of biomarker discovery in primary mouse neurons in vitro and validation in patient CSF from two independent large multicentre cohorts (EMIF-AD MBD, n = 310; PRIDE, n = 771).
RESULTS: First, we identify members of the protein disulfide isomerase (PDI) family in the neuronal UPR-activated secretome and validate secretion upon tau aggregation in vitro. Next, we demonstrate that PDIA1 and PDIA3 levels correlate with total- and phosphorylated-tau levels in CSF. PDIA1 levels are increased in CSF from AD patients compared to controls and patients with tau-unrelated frontotemporal and Lewy body dementia (LBD).
HIGHLIGHTS: Neuronal unfolded protein response (UPR) activation induces the secretion of protein disulfide isomerases (PDIs) in vitro. PDIA1 is secreted upon tau aggregation in neurons in vitro. PDIA1 and PDIA3 levels correlate with total and phosphorylated tau levels in CSF. PDIA1 levels are increased in CSF from Alzheimer's disease (AD) patients compared to controls. PDIA1 levels are not increased in CSF from tau-unrelated frontotemporal dementia (FTD) and Lewy body dementia (LBD) patients.

Keywords:  Alzheimer's disease; CSF biomarker; PDI; UPR; tau pathology

DOI:  https://doi.org/10.1002/alz.12978
bioRxiv. 2023 Feb 13. pii: 2023.02.11.528148. [Epub ahead of print]

The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction.

Wenjing Liang, Rachel Y Diao, Justin M Quiles, Rita H Najor, Liguo Chi, Benjamin P Woodall, Leonardo J Leon, Jason Duran, David M Cauvi, Antonio De Maio, Eric D Adler, Ã Sa B Gustafsson.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

DOI: https://doi.org/10.1101/2023.02.11.528148
FASEB J. 2023 Mar;37(3): e22816

Post-ovulatory aging is associated with altered patterns for small ubiquitin-like modifier (SUMO) proteins and SUMO-specific proteases.

Weber Beringui Feitosa, Patricia L Morris.

  Mammalian oocytes are ovulated arrested at metaphase of the second meiotic division. If they are not fertilized within a short period, the oocyte undergoes several progressive morphological, structural, and molecular changes during a process called oocyte aging. Herein, we focused on those functional events associated with proper cytoskeleton organization and those that correlate with spindle displacement and chromosome misalignment or scatter. Post-translational modifications by Small Ubiquitin-like Modifier (SUMO) proteins are involved in spindle organization and here we demonstrate that the SUMO pathway is involved in spindle morphology changes and chromosome movements during oocyte aging. SUMO-2/3 as well as the SUMO-specific proteases SENP-2 localization are affected by postovulatory aging in vitro. Consistent with these findings, UBC9 decreases during oocyte aging while differential ubiquitination patterns also correlate with in vitro oocyte aging. These results are consistent with postovulatory aging-related alterations in the posttranslational modifications of the spindle apparatus by SUMO and its SENP proteases. These findings are suggestive that such age-related changes in SUMOylation and the deSUMOylation of key target proteins in the spindle apparatus and kinetochore may be involved with spindle and chromosome alignment defects during mammalian oocyte postovulatory aging. Such findings may have implications for ART-related human oocyte aging in vitro regarding the activities of the SUMO pathway and fertilization success.

Keywords:  SUMOylation; aneuploidy; chromosome; spindle; ubiquitin

DOI:  https://doi.org/10.1096/fj.202200622R
Leukemia. 2023 Feb 22.

Human gene-engineered calreticulin mutant stem cells recapitulate MPN hallmarks and identify targetable vulnerabilities.

Johannes Foßelteder, Gabriel Pabst, Tommaso Sconocchia, Angelika Schlacher, Lisa Auinger, Karl Kashofer, Christine Beham-Schmid, Slave Trajanoski, Claudia Waskow, Wolfgang Schöll, Heinz Sill, Armin Zebisch, Albert Wölfler, Daniel Thomas, Andreas Reinisch.

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.

DOI: https://doi.org/10.1038/s41375-023-01848-6
Nat Rev Neurol. 2023 Feb 24.

Extracellular protein homeostasis in neurodegenerative diseases.

Mark R Wilson, Sandeep Satapathy, Michele Vendruscolo.

The protein homeostasis (proteostasis) system encompasses the cellular processes that regulate protein synthesis, folding, concentration, trafficking and degradation. In the case of intracellular proteostasis, the identity and nature of these processes have been extensively studied and are relatively well known. By contrast, the mechanisms of extracellular proteostasis are yet to be fully elucidated, although evidence is accumulating that their age-related progressive impairment might contribute to neuronal death in neurodegenerative diseases. Constitutively secreted extracellular chaperones are emerging as key players in processes that operate to protect neurons and other brain cells by neutralizing the toxicity of extracellular protein aggregates and promoting their safe clearance and disposal. Growing evidence indicates that these extracellular chaperones exert multiple effects to promote cell viability and protect neurons against pathologies arising from the misfolding and aggregation of proteins in the synaptic space and interstitial fluid. In this Review, we outline the current knowledge of the mechanisms of extracellular proteostasis linked to neurodegenerative diseases, and we examine the latest understanding of key molecules and processes that protect the brain from the pathological consequences of extracellular protein aggregation and proteotoxicity. Finally, we contemplate possible therapeutic opportunities for neurodegenerative diseases on the basis of this emerging knowledge.

DOI: https://doi.org/10.1038/s41582-023-00786-2
Proc Natl Acad Sci U S A. 2023 Feb 28. 120(9): e2214921120

Exploiting the intrinsic misfolding propensity of the KRAS oncoprotein.

Kobe Janssen, Filip Claes, Dido Van de Velde, Vanessa L Wehbi, Bert Houben, Yulia Lampi, Mieke Nys, Laleh Khodaparast, Ladan Khodaparast, Nikolaos Louros, Rob van der Kant, Joffre Verniers, Teresa Garcia, Meine Ramakers, Katerina Konstantoulea, Katerina Maragkou, Ramon Duran-Romaña, Mónica Musteanu, Mariano Barbacid, Bernard Scorneaux, Els Beirnaert, Joost Schymkowitz, Frederic Rousseau.

  Mutant KRAS is a major driver of oncogenesis in a multitude of cancers but remains a challenging target for classical small molecule drugs, motivating the exploration of alternative approaches. Here, we show that aggregation-prone regions (APRs) in the primary sequence of the oncoprotein constitute intrinsic vulnerabilities that can be exploited to misfold KRAS into protein aggregates. Conveniently, this propensity that is present in wild-type KRAS is increased in the common oncogenic mutations at positions 12 and 13. We show that synthetic peptides (Pept-ins™) derived from two distinct KRAS APRs could induce the misfolding and subsequent loss of function of oncogenic KRAS, both of recombinantly produced protein in solution, during cell-free translation and in cancer cells. The Pept-ins exerted antiproliferative activity against a range of mutant KRAS cell lines and abrogated tumor growth in a syngeneic lung adenocarcinoma mouse model driven by mutant KRAS G12V. These findings provide proof-of-concept that the intrinsic misfolding propensity of the KRAS oncoprotein can be exploited to cause its functional inactivation.

Keywords:  KRAS; oncogene; peptide; protein aggregation; protein folding

DOI:  https://doi.org/10.1073/pnas.2214921120
Neuro Oncol. 2023 Feb 20. pii: noad037. [Epub ahead of print]

Proteasomal pathway inhibition as a potential therapy for NF2-associated meningioma and schwannoma.

Srirupa Bhattacharyya, Janet L Oblinger, Roberta L Beauchamp, Zhenzhen Yin, Serkan Erdin, Priya Koundinya, Anna D Ware, Marc Ferrer, Justin T Jordan, Scott R Plotkin, Lei Xu, Long-Sheng Chang, Vijaya Ramesh.

  BACKGROUND: Neurofibromatosis 2 (NF2) is an inherited disorder caused by bi-allelic inactivation of the NF2 tumor suppressor gene. NF2-associated tumors, including schwannoma and meningioma, are resistant to chemotherapy, often recurring despite surgery and/or radiation, and have generally shown cytostatic response to signal transduction pathway inhibitors, highlighting the need for improved cytotoxic therapies.METHODS: Leveraging data from our previous high-throughput drug screening in NF2 preclinical models, we identified a class of compounds targeting the ubiquitin-proteasome pathway (UPP), and undertook studies using candidate UPP inhibitors, ixazomib/MLN9708, pevonedistat/MLN4924, and TAK-243/MLN7243. Employing human primary and immortalized meningioma (MN) cell lines, CRISPR-modified Schwann cells (SCs), and mouse Nf2 -/- SCs, we performed dose response testing, flow cytometry-based Annexin V and cell cycle analyses, and RNA-sequencing to identify potential underlying mechanism of apoptosis. In vivo efficacy was also assessed in orthotopic NF2-deficient meningioma and schwannoma tumor models.
RESULTS: Testing of three UPP inhibitors demonstrated potent reduction in cell viability and induction of apoptosis for ixazomib or TAK-243, but not pevonedistat. In vitro analyses revealed that ixazomib or TAK-243 downregulates expression of c-KIT and PDGFRα, as well as the E3 ubiquitin ligase SKP2 while upregulating genes associated with endoplasmic reticulum stress-mediated activation of the unfolded protein response (UPR). In vivo treatment of mouse models revealed delayed tumor growth, suggesting a therapeutic potential.
CONCLUSIONS: This study demonstrates the efficacy of proteasomal pathway inhibitors in meningioma and schwannoma preclinical models and lays the groundwork for use of these drugs as a promising novel treatment strategy for NF2 patients.

Keywords:  NF2; Ubiquitin-proteasome pathway inhibitors; apoptosis; meningioma; schwannoma

DOI:  https://doi.org/10.1093/neuonc/noad037
Curr Biol. 2023 Feb 22. pii: S0960-9822(23)00129-X. [Epub ahead of print]

Multiple ubiquitin E3 ligase genes antagonistically regulate chloroplast-associated protein degradation.

Sabri Mohd Ali, Na Li, Ziad Soufi, Jinrong Yao, Errin Johnson, Qihua Ling, R Paul Jarvis.

  The chloroplast is the most prominent member of a diverse group of plant organelles called the plastids, and it is characterized by its vital role in photosynthesis. 1,2,3 Most of the ∼3,000 different proteins in chloroplasts are synthesized in the cytosol in precursor (preprotein) form, each with a cleavable transit peptide. 4,5,6,7,8 Preproteins are imported via translocons in the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively. 9,10,11,12,13 Discovery of the chloroplast-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) demonstrated that the nucleocytosolic ubiquitin-proteasome system (UPS) targets the TOC apparatus to dynamically control protein import and chloroplast biogenesis in response to developmental and environmental cues. The relevant UPS pathway is termed chloroplast-associated protein degradation (CHLORAD). 14,15,16 Two homologs of SP1 exist, SP1-like1 (SPL1) and SPL2, but their roles have remained obscure. Here, we show that SP1 is ubiquitous in the Viridiplantae and that SPL2 and SPL1 appeared early during the evolution of the Viridiplantae and land plants, respectively. Through genetic and biochemical analysis, we reveal that SPL1 functions as a negative regulator of SP1, potentially by interfering with its ability to catalyze ubiquitination. In contrast, SPL2, the more distantly related SP1 homolog, displays partial functional redundancy with SP1. Both SPL1 and SPL2 modify the extent of leaf senescence, like SP1, but do so in diametrically opposite ways. Thus, SPL1 and SPL2 are bona fide CHLORAD system components with negative and positive regulatory functions that allow for nuanced control of this vital proteolytic pathway.

Keywords:  CHLORAD; E3 ligase; chloroplast; plastid; ubiquitin-proteasome system

DOI:  https://doi.org/10.1016/j.cub.2023.01.060
Curr Opin Struct Biol. 2023 Feb 15. pii: S0959-440X(23)00008-8. [Epub ahead of print]79 102534

Breaking free from the crystal lattice: Structural biology in solution to study protein degraders.

Kevin Haubrich, Valentina A Spiteri, William Farnaby, Frank Sobott, Alessio Ciulli.

Structural biology offers a versatile arsenal of techniques and methods to investigate the structure and conformational dynamics of proteins and their assemblies. The growing field of targeted protein degradation centres on the premise of developing small molecules, termed degraders, to induce proximity between an E3 ligase and a protein of interest to be signalled for degradation. This new drug modality brings with it new opportunities and challenges to structural biologists. Here we discuss how several structural biology techniques, including nuclear magnetic resonance, cryo-electron microscopy, structural mass spectrometry and small angle scattering, have been explored to complement X-ray crystallography in studying degraders and their ternary complexes. Together the studies covered in this review make a case for the invaluable perspectives that integrative structural biology techniques in solution can bring to understanding ternary complexes and designing degraders.

DOI: https://doi.org/10.1016/j.sbi.2023.102534
FEBS J. 2023 Feb 19.

Spatial position is a key determinant of N-glycan functionality of the scavenger receptor cysteine-rich domain of human hepsin.

Shijin Sun, Kaixuan Hu, Lina Wang, Meng Liu, Yikai Zhang, Ningzheng Dong, Qingyu Wu.

  The scavenger receptor cysteine-rich (SRCR) domain is a key constituent in diverse proteins. N-glycosylation is important in protein expression and function. In the SRCR domain of different proteins, N-glycosylation sites and functionality vary substantially. In this study, we examined the importance of N-glycosylation site positions in the SRCR domain of hepsin, a type II transmembrane serine protease involved in many pathophysiological processes. We analyzed hepsin mutants with alternative N-glycosylation sites in the SRCR and protease domains using three-dimensional modeling, site-directed mutagenesis, HepG2 cell expression, immunostaining, and western blotting. We found that the N-glycan function in the SRCR domain in promoting hepsin expression and activation on the cell surface cannot be replaced by alternatively created N-glycans in the protease domain. Within the SRCR domain, the presence of an N-glycan in a confined surface area was essential for calnexin-assisted protein folding, endoplasmic reticulum (ER) exiting, and zymogen activation of hepsin on the cell surface. Hepsin mutants with alternative N-glycosylation sites on the opposite side of the SRCR domain were trapped by ER chaperones, resulting in activation of the unfolded protein response in HepG2 cells. These results indicate that the spatial N-glycan positioning in the SRCR domain is a key determinant in the interaction with calnexin and subsequent cell surface expression of hepsin. These findings may help to understand the conservation and functionality of N-glycosylation sites in the SRCR domains of different proteins.

Keywords:  N-glycosylation; SRCR domain; calnexin; hepsin; type II transmembrane serine protease

DOI:  https://doi.org/10.1111/febs.16757
Nat Commun. 2023 Feb 17. 14(1): 918

Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria.

Narayan Prasad Parajuli, Andrew Emmerich, Chandra Sekhar Mandava, Michael Y Pavlov, Suparna Sanyal.

Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.

DOI: https://doi.org/10.1038/s41467-023-36528-7
Cell Death Differ. 2023 Feb 22.

The p53 endoplasmic reticulum stress-response pathway evolved in humans but not in mice via PERK-regulated p53 mRNA structures.

Leila Fusée, Norman Salomao, Anand Ponnuswamy, Lixiao Wang, Ignacio López, Sa Chen, Xiaolian Gu, Stavros Polyzoidis, Sivakumar Vadivel Gnanasundram, Robin Fahraeus.

Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.

DOI: https://doi.org/10.1038/s41418-023-01127-y
Nat Commun. 2023 Feb 20. 14(1): 948

Identification of global inhibitors of cellular glycosylation.

Daniel Madriz Sørensen, Christian Büll, Thomas D Madsen, Erandi Lira-Navarrete, Thomas Mandel Clausen, Alex E Clark, Aaron F Garretson, Richard Karlsson, Johan F A Pijnenborg, Xin Yin, Rebecca L Miller, Sumit K Chanda, Thomas J Boltje, Katrine T Schjoldager, Sergey Y Vakhrushev, Adnan Halim, Jeffrey D Esko, Aaron F Carlin, Ramon Hurtado-Guerrero, Roberto Weigert, Henrik Clausen, Yoshiki Narimatsu.

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

DOI: https://doi.org/10.1038/s41467-023-36598-7
Mol Syst Biol. 2023 Feb 23. e11544

Protein complexes in cells by AI-assisted structural proteomics.

Francis J O'Reilly, Andrea Graziadei, Christian Forbrig, Rica Bremenkamp, Kristine Charles, Swantje Lenz, Christoph Elfmann, Lutz Fischer, Jörg Stülke, Juri Rappsilber.

  Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry and co-fractionation mass spectrometry (CoFrac-MS) to identify protein-protein interactions in the model Gram-positive bacterium Bacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the SubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria.

Keywords:  AlphaFold-Multimer; crosslinking mass spectrometry; protein-protein interactions; pyruvate dehydrogenase; uncharacterized proteins

DOI:  https://doi.org/10.15252/msb.202311544
bioRxiv. 2023 Feb 18. pii: 2023.02.18.529068. [Epub ahead of print]

mRNA interactions with disordered regions control protein activity.

Yang Luo, Supriya Pratihar, Ellen H Horste, Sibylle Mitschka, Antonia S J S Mey, Hashim M Al-Hashimi, Christine Mayr.

The cytoplasm is compartmentalized into different translation environments. mRNAs use their 3â€²UTRs to localize to distinct cytoplasmic compartments, including TIS granules (TGs). Many transcription factors, including MYC, are translated in TGs. It was shown that translation of proteins in TGs enables the formation of protein complexes that cannot be established when these proteins are translated in the cytosol, but the mechanism is poorly understood. Here we show that MYC protein complexes that involve binding to the intrinsically disordered region (IDR) of MYC are only formed when MYC is translated in TGs. TG-dependent protein complexes require TG-enriched mRNAs for assembly. These mRNAs bind to a new and widespread RNA-binding domain in neutral or negatively charged IDRs in several transcription factors, including MYC. RNA-IDR interaction changes the conformational ensemble of the IDR, enabling the formation of MYC protein complexes that act in the nucleus and control functions that cannot be accomplished by cytosolically-translated MYC. We propose that certain mRNAs have IDR chaperone activity as they control IDR conformations. In addition to post-translational modifications, we found a novel mode of protein activity regulation. Since RNA-IDR interactions are prevalent, we suggest that mRNA-dependent control of protein functional states is widespread.

DOI: https://doi.org/10.1101/2023.02.18.529068
Cell Mol Gastroenterol Hepatol. 2023 Feb 17. pii: S2352-345X(23)00025-5. [Epub ahead of print]

Periostin protects against alcohol-related liver disease by activating autophagy by interacting with PDI.

Yanfei Zhang, Jiayu Jin, Heming Wu, Jingwen Huang, Shuting Ye, Jinhua Qiu, Gaoliang Ouyang, Tiantian Wu, Fan Liu, Yingfu Liu.

  BACKGROUND & AIMS: The matricellular protein periostin plays a critical role in liver inflammation, fibrosis, and even carcinoma. Here, the biological function of periostin in alcohol-related liver disease (ALD) was investigated.METHODS: We used wild-type (WT), Postn-null (Postn-/-) mice and Postn-/- mice with periostin recovery to investigate the biological function of periostin in ALD. Proximity-dependent biotin identification (BioID) analysis identified the protein that interacted with periostin, and coimmunoprecipitation (Co-IP) analysis validated the interaction between protein disulfide isomerase (PDI) and periostin. Pharmacological intervention and genetic knockdown of PDI were used to investigate the functional correlation between periostin and PDI in ALD development.
RESULTS: Periostin was markedly upregulated in the livers of mice that were fed ethanol. Interestingly, periostin deficiency severely aggravated ALD in mice, whereas the recovery of periostin in the livers of Postn-/- mice significantly ameliorated ALD. Mechanistic studies showed that the upregulation of periostin alleviated ALD by activating autophagy through inhibition of the mTORC1 pathway, which was verified in murine models treated with the mTOR inhibitor rapamycin and the autophagy inhibitor MHY1485. Furthermore, a protein interaction map of periostin was generated by BioID analysis. Interaction profile analysis identified PDI as a key protein that interacted with periostin. Intriguingly, periostin-mediated enhancement of autophagy by inhibiting the mTORC1 pathway in ALD depended on its interaction with PDI. Moreover, alcohol-induced periostin overexpression was regulated by transcription factor EB (TFEB).
CONCLUSIONS: Collectively, these findings clarify a novel biological function and mechanism of periostin in ALD and the periostin-PDI-mTORC1 axis is a critical determinant of ALD.

Keywords:  ALD; BioID; Hepatic steatosis; PDI; Periostin

DOI:  https://doi.org/10.1016/j.jcmgh.2023.02.005