bims-proteo 2024-02-04 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–02–04
28 papers selected by
Eric Chevet, INSERM

The p24-family and COPII subunit SEC24C facilitate the clearance of alpha1-antitrypsin Z from the endoplasmic reticulum to lysosomes.
Stress response silencing by an E3 ligase mutated in neurodegeneration.
ByeTAC: Bypassing an E3 Ligase for Targeted Protein Degradation.
Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions.
The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration.
"Sticki-ER": functions of the platelet endoplasmic reticulum.
Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation.
GPI-anchored Gas1 protein regulates cytosolic proteostasis in budding yeast.
Multiscale In Silico Study of the Mechanism of Activation of the RtcB Ligase by the PTP1B Phosphatase.
Establishing Order through Disorder by the Hsp90 Molecular Chaperone.
Cellular targets and lysine selectivity of the HERC5 ISG15 ligase.
Systematic identification of 20S proteasome substrates.
Protocol for the quantitative identification of endogenously ISGylated proteins from mammalian cell lines.
Mitochondrial protein synthesis quality control.
N-glycosylation as a eukaryotic protective mechanism against protein aggregation.
Recapitulating and reversing human brain ribosomopathy defects via the maladaptive integrated stress response.
Calcium Flow at ER-TGN Contact Sites Facilitates Secretory Cargo Export.
The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A.
A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle.
Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins.
A close-up shot of protein-protein docking, from experiment to theory and reverse with the PROTAC performers.
Expanding the ligandable proteome by paralog hopping with covalent probes.
Sirtuin3 promotes the degradation of hepatic Z alpha-1 antitrypsin through lipophagy.
Folding-upon-binding pathways of an intrinsically disordered protein from a deep Markov state model.
Improving Split Reporters of Protein-Protein Interactions through Orthology-Based Protein Engineering.
A widespread bacterial protein compartment sequesters and stores elemental sulfur.
DPCD is a regulator of R2TP in ciliogenesis initiation through Akt signaling.
Cell competition and the regulation of protein homeostasis.

Mol Biol Cell. 2024 Jan 31. mbcE23060257

The p24-family and COPII subunit SEC24C facilitate the clearance of alpha1-antitrypsin Z from the endoplasmic reticulum to lysosomes.

Benjamin S Roberts, Debashree Mitra, Sudhanshu Abishek, Richa Beher, Prasanna Satpute-Krishnan.

A subpopulation of the alpha-1-antitrypsin misfolding Z mutant (ATZ) is cleared from the endoplasmic reticulum (ER) via an ER-to-lysosome-associated degradation (ERLAD) pathway. Here, we report that the COPII subunit SEC24C and the p24-family of proteins facilitate the clearance of ATZ via ERLAD. In addition to the previously reported ERLAD components calnexin and FAM134B, we discovered that ATZ co-immunoprecipitates with the p24-family members TMP21 and TMED9. This contrasts with wild type alpha1-antitrypsin, which did not co-immunoprecipitate with FAM134B, calnexin or the p24-family members. Live-cell imaging revealed that ATZ and the p24-family members traffic together from the ER to lysosomes. Using chemical inhibitors to block ER exit or autophagy, we demonstrated that p24-family members and ATZ co-accumulate at SEC24C marked ER-exit sites or in ER-derived compartments, respectively. Furthermore, depletion of SEC24C, TMP21 or TMED9 inhibited lysosomal trafficking of ATZ and resulted in the increase of intracellular ATZ levels. Conversely, overexpression of these p24-family members resulted in the reduction of ATZ levels. Intriguingly, the p24-family members co-immunoprecipitate with ATZ, FAM134B and SEC24C. Thus, we propose a model in which the p24-family functions in an adaptor complex linking SEC24C with the ERLAD machinery for the clearance of ATZ. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E23-06-0257
Nature. 2024 Jan 31.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Diane L Haakonsen, Michael Heider, Andrew J Ingersoll, Kayla Vodehnal, Samuel R Witus, Takeshi Uenaka, Marius Wernig, Michael Rapé.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

DOI: https://doi.org/10.1038/s41586-023-06985-7
bioRxiv. 2024 Jan 21. pii: 2024.01.20.576376. [Epub ahead of print]

ByeTAC: Bypassing an E3 Ligase for Targeted Protein Degradation.

Eslam M H Ali, Cody A Loy, Darci J Trader.

Targeted protein degradation utilizing a bifunctional molecule to initiate ubiquitination and subsequent degradation by the 26S proteasome has been shown to be a powerful therapeutic intervention. Many bifunctional molecules, including covalent and non-covalent ligands to proteins of interest, have been developed. The traditional target protein degradation methodology targets the protein of interest in both healthy and diseased cell populations, and a therapeutic window is obtained based on the overexpression of the targeted protein. We report here a series of bifunctional degraders that do not rely on interacting with an E3 ligase, but rather a 26S proteasome subunit, which we have named ByeTACs: Bypassing E3 Targeting Chimeras. Rpn-13 is a non-essential ubiquitin receptor for the 26S proteasome. Cells under significant stress or require significant ubiquitin-dependent degradation of proteins for survival, incorporate Rpn-13 in the 26S to increase protein degradation rates. The targeted protein degraders reported here are bifunctional molecules that include a ligand to Rpn-13 and BRD4, the protein of interest we wish to degrade. We synthesized a suite of degraders with varying PEG chain lengths and showed that bifunctional molecules that incorporate a Rpn-13 binder (TCL1) and a BRD4 binder (JQ1) with a PEG linker of 3 or 4 units are the most effective to induce BRD4 degradation. We also demonstrate that our new targeted protein degraders are dependent upon proteasome activity and Rpn-13 expression levels. This establishes a new mechanism of action for our ByeTACs that can be employed for the targeted degradation of a wide variety of protein substrates.

DOI: https://doi.org/10.1101/2024.01.20.576376
Nucleic Acids Res. 2024 Jan 28. pii: gkae006. [Epub ahead of print]

Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions.

Jagannath Misra, Kenneth R Carlson, Dan F Spandau, Ronald C Wek.

Diverse environmental insults induce the integrated stress response (ISR), which features eIF2 phosphorylation and translational control that serves to restore protein homeostasis. The eIF2 kinase GCN2 is a first responder in the ISR that is activated by amino acid depletion and other stresses not directly related to nutrients. Two mechanisms are suggested to trigger an ordered process of GCN2 activation during stress: GCN2 monitoring stress via accumulating uncharged tRNAs or by stalled and colliding ribosomes. Our results suggest that while ribosomal collisions are indeed essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 via its direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. GCN2 activation by UV irradiation features lowered amino acids and increased uncharged tRNAs and UV-induced ribosome collisions are suggested to be dispensable. We conclude that there are multiple mechanisms that activate GCN2 during diverse stresses.

DOI: https://doi.org/10.1093/nar/gkae006
J Biol Chem. 2024 Jan 30. pii: S0021-9258(24)00069-3. [Epub ahead of print] 105693

The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration.

Jasper Weinberg, Elizabeth Whitcomb, Andrew Bohm, Uday Kumar Chekkilla, Allen Taylor.

  Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293 and HELA cells. But the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1, but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Keywords:  SMURF1; UBCH7 (UBE2L3); cell mobility; development; ligase; p27; ubiquitin conjugating enzyme

DOI:  https://doi.org/10.1016/j.jbc.2024.105693
Antioxid Redox Signal. 2024 Jan 29.

"Sticki-ER": functions of the platelet endoplasmic reticulum.

Yvonne Kong, Joyce Chiu, Freda H Passam.

SIGNIFICANCE: Platelets are the smallest cells in circulation (measuring 2 microns in diameter). Platelet activation is necessary for thrombus formation and relies on calcium mobilisation from the endoplasmic reticulum (ER). Platelet activation leads to mobilization of ER resident proteins to the platelet surface; these are essential for the function of platelet receptors and intercellular interactions.
RECENT ADVANCES: ER homeostasis is maintained by an appropriate redox balance, regulated calcium stores and normal protein folding. Disruption of platelet ER homeostasis can cause ER stress which contributes to platelet activation. ER stress results in the externalisation of ER proteins to the cell surface, including members of the protein disulfide isomerase family and chaperones.
CRITICAL ISSUES: The ER is central to platelet function, but our understanding of its regulation is incomplete. Previous studies have focused on the function of platelet protein disulfide isomerase family members in the extracellular space, and much less on their intracellular role. How platelets maintain ER homeostasis and how they direct ER chaperone proteins to facilitate intercellular signalling is unknown.
FUTURE DIRECTIONS: An understanding of ER functions in the platelet is essential as these may determine critical platelet activities such as secretion and adhesion. Studies are necessary to understand the redox reactions of platelet disulfide isomerases in the intracellular versus extracellular space, as these differentially affect platelet function. Unresolved questions are how platelet ER proteins control calcium release and protein folding in response to ER stress. Targeting the platelet ER may have therapeutic application in metabolic and neoplastic disease.

DOI: https://doi.org/10.1089/ars.2024.0566
ACS Chem Biol. 2024 Feb 02.

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

Seong Ho Hong, Anand Divakaran, Akane Osa, Oscar W Huang, 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 the 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/10.1021/acschembio.3c00642
G3 (Bethesda). 2024 Jan 30. pii: jkad263. [Epub ahead of print]

GPI-anchored Gas1 protein regulates cytosolic proteostasis in budding yeast.

Yuhao Wang, Linhao Ruan, Rong Li.

  The decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation through the "mitochondrial as guardian in cytosol" (MAGIC) pathway. Here, we report an unexpected role of Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase in the budding yeast, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of GAS1 inhibits MAGIC but elevates protein ubiquitination and UPS-mediated protein degradation. Interestingly, we found that the Gas1 protein exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins through MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1-E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.

Keywords:  GPI; Gas1; MAGIC; budding yeast; mitochondria; proteostasis

DOI:  https://doi.org/10.1093/g3journal/jkad263
J Chem Inf Model. 2024 Jan 29.

Multiscale In Silico Study of the Mechanism of Activation of the RtcB Ligase by the PTP1B Phosphatase.

Sayyed Jalil Mahdizadeh, Michael Stier, Antonio Carlesso, Aurore Lamy, Melissa Thomas, Leif A Eriksson.

Inositol-requiring enzyme 1 (IRE1) is a transmembrane sensor that is part of a trio of sensors responsible for controlling the unfolded protein response within the endoplasmic reticulum (ER). Upon the accumulation of unfolded or misfolded proteins in the ER, IRE1 becomes activated and initiates the cleavage of a 26-nucleotide intron from human X-box-containing protein 1 (XBP1). The cleavage is mediated by the RtcB ligase enzyme, which splices together two exons, resulting in the formation of the spliced isoform XBP1s. The XBP1s isoform activates the transcription of genes involved in ER-associated degradation to maintain cellular homeostasis. The catalytic activity of RtcB is controlled by the phosphorylation and dephosphorylation of three tyrosine residues (Y306, Y316, and Y475), which are regulated by the ABL1 tyrosine kinase and PTP1B phosphatase, respectively. This study focuses on investigating the mechanism by which the PTP1B phosphatase activates the RtcB ligase using a range of advanced in silico methods. Protein-protein docking identified key interacting residues between RtcB and PTP1B. Notably, the phosphorylated Tyr306 formed hydrogen bonds and salt bridge interactions with the "gatekeeper" residues Arg47 and Lys120 of the inactive PTP1B. Classical molecular dynamics simulation emphasized the crucial role of Asp181 in the activation of PTP1B, driving the conformational change from an open to a closed state of the WPD-loop. Furthermore, QM/MM-MD simulations provided insights into the free energy landscape of the dephosphorylation reaction mechanism of RtcB, which is mediated by the PTP1B phosphatase.

DOI: https://doi.org/10.1021/acs.jcim.3c01600
J Mol Biol. 2024 Jan 30. pii: S0022-2836(24)00026-3. [Epub ahead of print] 168460

Establishing Order through Disorder by the Hsp90 Molecular Chaperone.

Neethu Babu, Brian C Freeman.

The Heat Shock Protein 90 (Hsp90) molecular chaperone is a key driver of protein homeostasis (proteostasis) under physiologically normal and stress conditions. In eukaryotes, Hsp90 is essential and is one of the most abundant proteins in a cell where the chaperone shuttles between the cytoplasm and nucleus to fold, stabilize, and regulate client proteins and protein complexes. Numerous high-throughput screens have mapped the Hsp90 interactome, building a vast network comprising ∼25% of the proteome in budding yeast. How Hsp90 is able to associate with this diverse and large cadre of targets is critical to comprehending how the proteostatic process works. Here, we review recent progress on our understanding of the molecular underpinnings driving Hsp90-client interactions from both the perspective of the targets and Hsp90. In addition to considering the available Hsp90-client structures, we also assessed recently identified Hsp90-client peptide complexes to build a model that justifies how Hsp90 might recognize a wide spectrum of target proteins. In brief, Hsp90 either directly recognizes a site within an intrinsically disordered region (IDR) of a client protein to transiently regulate that client or it associates with an unstructured polypeptide section created by the concerted efforts of multiple chaperones and cochaperones to stably associate with a client. Overall, Hsp90 exploits a common recognition property (i.e., IDR) within diverse clients to support chaperone-actionthereby enabling its central role in proteostasis.

DOI: https://doi.org/10.1016/j.jmb.2024.168460
iScience. 2024 Feb 16. 27(2): 108820

Cellular targets and lysine selectivity of the HERC5 ISG15 ligase.

Xu Zhao, Jessica M Perez, Peter A Faull, Catherine Chan, Femke W Munting, Larissa A Canadeo, Can Cenik, Jon M Huibregtse.

  ISG15 is a type I interferon-induced ubiquitin-like modifier that functions in innate immune responses. The major human ISG15 ligase is hHERC5, a ribosome-associated HECT E3 that broadly ISGylates proteins cotranslationally. Here, we characterized the hHERC5-dependent ISGylome and identified over 2,000 modified lysines in over 1,100 proteins in IFN-β-stimulated cells. In parallel, we compared the substrate selectivity hHERC5 to the major mouse ISG15 ligase, mHERC6, and analysis of sequences surrounding ISGylation sites revealed that hHERC5 and mHERC6 have distinct preferences for amino acid sequence context. Several features of the datasets were consistent with ISGylation of ribosome-tethered nascent chains, and mHERC6, like hHERC5, cotranslationally modified nascent polypeptides. The ISGylome datasets presented here represent the largest numbers of protein targets and modification sites attributable to a single Ub/Ubl ligase and the lysine selectivities of the hHERC5 and mHERC6 enzymes may have implications for the activities of HECT domain ligases, generally.

Keywords:  Biochemistry; Immunology; Molecular biology; Transcriptomics

DOI:  https://doi.org/10.1016/j.isci.2024.108820
Mol Syst Biol. 2024 Jan 29.

Systematic identification of 20S proteasome substrates.

Monika Pepelnjak, Rivkah Rogawski, Galina Arkind, Yegor Leushkin, Irit Fainer, Gili Ben-Nissan, Paola Picotti, Michal Sharon.

  For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology.

Keywords:  20S Proteasome; Intrinsically Disordered Proteins; Mass Spectrometry; Protein Degradation; Proteolytic Processing

DOI:  https://doi.org/10.1038/s44320-024-00015-y
STAR Protoc. 2024 Jan 29. pii: S2666-1667(24)00008-X. [Epub ahead of print]5(1): 102843

Protocol for the quantitative identification of endogenously ISGylated proteins from mammalian cell lines.

Christopher P Wardlaw, Matthew M Miele, Zhuoning Li, Ronald C Hendrickson, John H J Petrini.

  Ubiquitin-like protein ISG15 plays an important role in an array of cellular functions via its covalent attachment to target proteins (ISGylation). Here, we present a protocol for the identification of ISGylated proteins that avoids the caveats associated with ISG15 overexpression and minimizes the likelihood of false positives. We describe steps for the tagging of endogenous ISG15, followed by genotyping and clone selection. We then detail steps for ISGylation induction, the isolation of ISGylated proteins, and their identification via quantitative mass spectrometry. For complete details on the use and execution of this protocol, please refer to Wardlaw and Petrini.1.

Keywords:  CRISPR; Cell Biology; Mass Spectrometry; Molecular Biology; Proteomics

DOI:  https://doi.org/10.1016/j.xpro.2024.102843
Hum Mol Genet. 2024 Jan 27. pii: ddae012. [Epub ahead of print]

Mitochondrial protein synthesis quality control.

Lidiia Koludarova, Brendan J Battersby.

  Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical barrier to separate transcription and translation, nor is there evidence that quality control surveillance pathways are active to prevent translation on faulty mRNA transcripts. Mitochondrial ribosomes synthesize 13 hydrophobic proteins that require co-translational insertion into the inner membrane of the organelle. To maintain the integrity of the inner membrane, which is essential for organelle function, requires responsive quality control mechanisms to recognize aberrations in protein synthesis. In this review, we explore how defects in mitochondrial protein synthesis can arise due to the culmination of inherent mistakes that occur throughout the steps of gene expression. In turn, we examine the stepwise series of quality control processes that are needed to eliminate any mistakes that would perturb organelle homeostasis. We aim to provide an integrated view on the quality control mechanisms of mitochondrial protein synthesis and to identify promising avenues for future research.

Keywords:  AFG3L2; MTRFR; OMA1; OPA1; OXA1L; RNA processing; cell stress; co-translational quality control; fusion open reading frames; membrane morphology; mitochondria; non-stop mRNA; post-transcriptional; protein synthesis; proteostasis; ribosome quality control; ribosomes

DOI:  https://doi.org/10.1093/hmg/ddae012
Sci Adv. 2024 Feb 02. 10(5): eadk8173

N-glycosylation as a eukaryotic protective mechanism against protein aggregation.

Ramon Duran-Romaña, Bert Houben, Matthias De Vleeschouwer, Nikolaos Louros, Matthew P Wilson, Gert Matthijs, Joost Schymkowitz, Frederic Rousseau.

The tendency for proteins to form aggregates is an inherent part of every proteome and arises from the self-assembly of short protein segments called aggregation-prone regions (APRs). While posttranslational modifications (PTMs) have been implicated in modulating protein aggregation, their direct role in APRs remains poorly understood. In this study, we used a combination of proteome-wide computational analyses and biophysical techniques to investigate the potential involvement of PTMs in aggregation regulation. Our findings reveal that while most PTM types are disfavored near APRs, N-glycosylation is enriched and evolutionarily selected, especially in proteins prone to misfolding. Experimentally, we show that N-glycosylation inhibits the aggregation of peptides in vitro through steric hindrance. Moreover, mining existing proteomics data, we find that the loss of N-glycans at the flanks of APRs leads to specific protein aggregation in Neuro2a cells. Our findings indicate that, among its many molecular functions, N-glycosylation directly prevents protein aggregation in higher eukaryotes.

DOI: https://doi.org/10.1126/sciadv.adk8173
Sci Adv. 2024 Feb 02. 10(5): eadk1034

Recapitulating and reversing human brain ribosomopathy defects via the maladaptive integrated stress response.

Wei Zhang, Minjie Zhang, Li Ma, Supawadee Jariyasakulroj, Qing Chang, Ziying Lin, Zhipeng Lu, Jian-Fu Chen.

Animal or human models recapitulating brain ribosomopathies are incomplete, hampering development of urgently needed therapies. Here, we generated genetic mouse and human cerebral organoid models of brain ribosomopathies, caused by mutations in small nucleolar RNA (snoRNA) SNORD118. Both models exhibited protein synthesis loss, proteotoxic stress, and p53 activation and led to decreased proliferation and increased death of neural progenitor cells (NPCs), resulting in brain growth retardation, recapitulating features in human patients. Loss of SNORD118 function resulted in an aberrant upregulation of p-eIF2α, the mediator of integrated stress response (ISR). Using human iPSC cell-based screen, we identified small-molecule 2BAct, an ISR inhibitor, which potently reverses mutant NPC defects. Targeting ISR by 2BAct mitigated ribosomopathy defects in both cerebral organoid and mouse models. Thus, our SNORD118 mutant organoid and mice recapitulate human brain ribosomopathies and cross-validate maladaptive ISR as a key disease-driving mechanism, pointing to a therapeutic intervention strategy.

DOI: https://doi.org/10.1126/sciadv.adk1034
Mol Biol Cell. 2024 Jan 31. mbcE23030099

Calcium Flow at ER-TGN Contact Sites Facilitates Secretory Cargo Export.

Bulat R Ramazanov, Anup Parchure, Rosaria Di Martino, Abhishek Kumar, Minhwan Chung, Yeongho Kim, Oliver Griesbeck, Martin A Schwartz, Alberto Luini, Julia von Blume.

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), allowing protein-mediated exchange of molecular species such as lipids. Here, we show that the TGN export of secretory proteins requires the integrity of ER-TGN MCS and inositol 3 phosphate receptor (IP3R)-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real time. These data show that ER-TGN MCS facilitates the Ca2+ transfer required for Ca2+-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.

DOI: https://doi.org/10.1091/mbc.E23-03-0099
Nat Struct Mol Biol. 2024 Jan 29.

The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A.

Jailson Brito Querido, Masaaki Sokabe, Irene Díaz-López, Yuliya Gordiyenko, Christopher S Fraser, V Ramakrishnan.

Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.

DOI: https://doi.org/10.1038/s41594-023-01196-0
Nat Commun. 2024 Feb 02. 15(1): 1007

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle.

Junsun Park, Hyunmin Kim, Daniel Gestaut, Seyeon Lim, Kwadwo A Opoku-Nsiah, Alexander Leitner, Judith Frydman, Soung-Hun Roh.

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. PhLP2A binds to open apo-TRiC through polyvalent domain-specific contacts with its chamber's equatorial and apical regions. PhLP2A N-terminal H3-domain binding to subunits CCT3/4 apical domains displace PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to positively charged inner surface residues from CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

DOI: https://doi.org/10.1038/s41467-024-45242-x
EMBO Rep. 2024 Jan 29.

Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins.

Janika Schmitt, Emma Poole, Ian Groves, David J Owen, Stephen C Graham, John Sinclair, Bernard T Kelly.

  The exquisite specificity of antibodies can be harnessed to effect targeted degradation of membrane proteins. Here, we demonstrate targeted protein removal utilising a protein degradation domain derived from the endogenous human protein Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). Recombinant antibodies genetically fused to this domain drive the degradation of membrane proteins that undergo constitutive internalisation and recycling, including the transferrin receptor and the human cytomegalovirus latency-associated protein US28. We term this approach PACTAC (PCSK9-Antibody Clearance-Targeting Chimeras).

Keywords:  Human Cytomegalovirus; PACTAC; PCSK9; Targeted Protein Degradation

DOI:  https://doi.org/10.1038/s44319-024-00063-3
J Biomol Struct Dyn. 2024 Jan 29. 1-8

A close-up shot of protein-protein docking, from experiment to theory and reverse with the PROTAC performers.

Khanh Quynh Thi Nguyen, Hieu Hien Nguyen, Huong Thi Thu Phung, Khanh Linh Chung, Thien Y Vu.

  PROTACs (Proteolysis Targeting Chimeras), heterobifunctional molecules, exhibit selectivity in degrading target proteins through E3 ubiquitin ligases. Designing effective PROTACs requires a deep understanding of the intricate binding interactions in the ternary complex (POI/PROTAC/E3 ligase), crucial for efficient target protein degradation. To address this challenge, we introduce a novel computational virtual screening method that considers essential amino acid interactions between the protein of interest and the chosen E3 ligase. This approach enhances accuracy and reliability, facilitating the strategic development of potent PROTACs. Utilizing a crystallized model of the VHL:PROTAC:SMARCA2BD ternary complex (PDB: 7Z6L), we assessed the effectiveness of our method. Our study reveals that increasing the number of essential restraints between the two proteins reduces the generated docking poses, leading to closer alignment with the experimental ternary complex. Specifically, utilizing three restraints showed the closest resemblance to the published complex, highlighting crucial interactions such as an H-bond between A:Gln 89 and B:Asn 67, along with two hydrophobic interactions: A:Gly 22 with B:Arg 69 and A:Glu 37 with B:Pro 99. This resulted in a significant decrease in the mean RMSD value from 31.8 and 31.0 Å to 24.4 Å, respectively. This underscores the importance of incorporating multiple essential restraints to enhance docking accuracy. Building on this progress, we introduce a systematic approach to design potential PROTACs between the Estrogen receptor and the E3 ligase, utilizing bridging intermediates with 4, 6, or 7 carbon atoms. By providing a more accurate and efficient means of identifying optimal PROTAC candidates, this approach has the potential to accelerate the development of targeted therapies and reduce the time and costs associated with drug discovery.Communicated by Ramaswamy H. Sarma.

Keywords:  Desmond; E3 ligase; PIPER; PROTAC building; Protein – Protein docking; Schrödinger suites; bioluminate; estrogen receptor

DOI:  https://doi.org/10.1080/07391102.2024.2308778
bioRxiv. 2024 Jan 19. pii: 2024.01.18.576274. [Epub ahead of print]

Expanding the ligandable proteome by paralog hopping with covalent probes.

Yuanjin Zhang, Zhonglin Liu, Marsha Hirschi, Oleg Brodsky, Eric Johnson, Sang Joon Won, Asako Nagata, Matthew D Petroski, Jaimeen D Majmudar, Sherry Niessen, Todd VanArsdale, Adam M Gilbert, Matthew M Hayward, Al E Stewart, Andrew R Nager, Bruno Melillo, Benjamin Cravatt.

More than half of the ∼20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

DOI: https://doi.org/10.1101/2024.01.18.576274
Hepatol Commun. 2024 Feb 01. pii: e0370. [Epub ahead of print]8(2):

Sirtuin3 promotes the degradation of hepatic Z alpha-1 antitrypsin through lipophagy.

Brittney Poole, Regina Oshins, Zhiguang Huo, Alek Aranyos, Jesse West, Sergio Duarte, Virginia C Clark, Thiago Beduschi, Ali Zarrinpar, Mark Brantly, Nazli Khodayari.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by misfolding and accumulation of mutant alpha-1 antitrypsin (ZAAT) in the endoplasmic reticulum of hepatocytes. Hepatic ZAAT aggregates acquire a toxic gain-of-function that impacts the endoplasmic reticulum which is theorized to cause liver disease in individuals with AATD who present asymptomatic until late-stage cirrhosis. Currently, there is no treatment for AATD-mediated liver disease except liver transplantation. In our study of mitochondrial RNA, we identified that Sirtuin3 (SIRT3) plays a role in the hepatic phenotype of AATD.
METHODS: Utilizing RNA and protein analysis in an in vitro AATD model, we investigated the role of SIRT3 in the pathophysiology of AATD-mediated liver disease while also characterizing our novel, transgenic AATD mouse model.
RESULTS: We show lower expression of SIRT3 in ZAAT-expressing hepatocytes. In contrast, the overexpression of SIRT3 increases hepatic ZAAT degradation. ZAAT degradation mediated by SIRT3 appeared independent of proteasomal degradation and regular autophagy pathways. We observed that ZAAT-expressing hepatocytes have aberrant accumulation of lipid droplets, with ZAAT polymers localizing on the lipid droplet surface in a direct interaction with Perilipin2, which coats intracellular lipid droplets. SIRT3 overexpression also induced the degradation of lipid droplets in ZAAT-expressing hepatocytes. We observed that SIRT3 overexpression induces lipophagy by enhancing the interaction of Perilipin2 with HSC70. ZAAT polymers then degrade as a consequence of the mobilization of lipids through this process.
CONCLUSIONS: In this context, SIRT3 activation may eliminate the hepatic toxic gain-of-function associated with the polymerization of ZAAT, providing a rationale for a potential novel therapeutic approach to the treatment of AATD-mediated liver disease.

DOI: https://doi.org/10.1097/HC9.0000000000000370
Proc Natl Acad Sci U S A. 2024 Feb 06. 121(6): e2313360121

Folding-upon-binding pathways of an intrinsically disordered protein from a deep Markov state model.

Thomas R Sisk, Paul Robustelli.

  A central challenge in the study of intrinsically disordered proteins is the characterization of the mechanisms by which they bind their physiological interaction partners. Here, we utilize a deep learning-based Markov state modeling approach to characterize the folding-upon-binding pathways observed in a long timescale molecular dynamics simulation of a disordered region of the measles virus nucleoprotein NTAIL reversibly binding the X domain of the measles virus phosphoprotein complex. We find that folding-upon-binding predominantly occurs via two distinct encounter complexes that are differentiated by the binding orientation, helical content, and conformational heterogeneity of NTAIL. We observe that folding-upon-binding predominantly proceeds through a multi-step induced fit mechanism with several intermediates and do not find evidence for the existence of canonical conformational selection pathways. We observe four kinetically separated native-like bound states that interconvert on timescales of eighty to five hundred nanoseconds. These bound states share a core set of native intermolecular contacts and stable NTAIL helices and are differentiated by a sequential formation of native and non-native contacts and additional helical turns. Our analyses provide an atomic resolution structural description of intermediate states in a folding-upon-binding pathway and elucidate the nature of the kinetic barriers between metastable states in a dynamic and heterogenous, or "fuzzy", protein complex.

Keywords:  Markov state models; deep learning; intrinsically disordered proteins; molecular dynamics; protein folding

DOI:  https://doi.org/10.1073/pnas.2313360121
ACS Chem Biol. 2024 Jan 30.

Improving Split Reporters of Protein-Protein Interactions through Orthology-Based Protein Engineering.

Louise-Marie Rakotoarison, Alison G Tebo, Dorothea Böken, Stephanie Board, Lina El Hajji, Arnaud Gautier.

Protein-protein interactions (PPIs) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of splitFAST2, an improved reporter displaying higher brightness, lower self-complementation, and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, splitFAST2 is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

DOI: https://doi.org/10.1021/acschembio.3c00631
Sci Adv. 2024 Feb 02. 10(5): eadk9345

A widespread bacterial protein compartment sequesters and stores elemental sulfur.

Robert Benisch, Michael P Andreas, Tobias W Giessen.

Subcellular compartments often serve to store nutrients or sequester labile or toxic compounds. As bacteria mostly do not possess membrane-bound organelles, they often have to rely on protein-based compartments. Encapsulins are one of the most prevalent protein-based compartmentalization strategies found in prokaryotes. Here, we show that desulfurase encapsulins can sequester and store large amounts of crystalline elemental sulfur. We determine the 1.78-angstrom cryo-EM structure of a 24-nanometer desulfurase-loaded encapsulin. Elemental sulfur crystals can be formed inside the encapsulin shell in a desulfurase-dependent manner with l-cysteine as the sulfur donor. Sulfur accumulation can be influenced by the concentration and type of sulfur source in growth medium. The selectively permeable protein shell allows the storage of redox-labile elemental sulfur by excluding cellular reducing agents, while encapsulation substantially improves desulfurase activity and stability. These findings represent an example of a protein compartment able to accumulate and store elemental sulfur.

DOI: https://doi.org/10.1126/sciadv.adk9345
Cell Rep. 2024 Jan 31. pii: S2211-1247(24)00041-X. [Epub ahead of print]43(2): 113713

DPCD is a regulator of R2TP in ciliogenesis initiation through Akt signaling.

Yu-Qian Mao, Thiago V Seraphim, Yimei Wan, Ruikai Wu, Etienne Coyaud, Muhammad Bin Munim, Antonio Mollica, Estelle Laurent, Mohan Babu, Vito Mennella, Brian Raught, Walid A Houry.

  R2TP is a chaperone complex consisting of the AAA+ ATPases RUVBL1 and RUVBL2, as well as RPAP3 and PIH1D1 proteins. R2TP is responsible for the assembly of macromolecular complexes mainly acting through different adaptors. Using proximity-labeling mass spectrometry, we identified deleted in primary ciliary dyskinesia (DPCD) as an adaptor of R2TP. Here, we demonstrate that R2TP-DPCD influences ciliogenesis initiation through a unique mechanism by interaction with Akt kinase to regulate its phosphorylation levels rather than its stability. We further show that DPCD is a heart-shaped monomeric protein with two domains. A highly conserved region in the cysteine- and histidine-rich domains-containing proteins and SGT1 (CS) domain of DPCD interacts with the RUVBL2 DII domain with high affinity to form a stable R2TP-DPCD complex both in cellulo and in vitro. Considering that DPCD is one among several CS-domain-containing proteins found to associate with RUVBL1/2, we propose that RUVBL1/2 are CS-domain-binding proteins that regulate complex assembly and downstream signaling.

Keywords:  AAA+ ATPases; Akt; CP: Cell biology; CS domain; DPCD; R2TP; ciliogenesis; molecular chaperone

DOI:  https://doi.org/10.1016/j.celrep.2024.113713
Curr Opin Cell Biol. 2024 Jan 31. pii: S0955-0674(24)00002-4. [Epub ahead of print]87 102323

Cell competition and the regulation of protein homeostasis.

Shruthi Krishnan, Pranab K Paul, Tristan A Rodriguez.

  The process of embryonic development involves remarkable cellular plasticity, which governs the coordination between cells necessary to build an organism. One role of this plasticity is to ensure that when aberrant cells are eliminated, growth adjustment occurs so that the size of the tissue is maintained. An important regulator of cellular plasticity that ensures cellular cooperation is a fitness-sensing mechanism termed cell competition. During cell competition, cells with defects that lower fitness but do not affect viability, such as those that cause impaired signal transduction, slower cellular growth, mitochondrial dysregulation or impaired protein homeostasis, are killed when surrounded by fitter cells. This is accompanied by the compensatory proliferation of the surviving cells. The underlying factors and mechanisms that demarcate certain cells as less fit than their neighbouring cells and losers of cell competition are still relatively unknown. Recent evidence has pointed to mitochondrial defects and proteotoxic stress as important hallmarks of these loser cells. Here, we review recent advances in this area, focussing on the role of mitochondrial activity and protein homeostasis as major mechanisms determining competitive cell fitness during development and the importance of cell proteostasis in determining cell fitness.

Keywords:  Cell competition; Cell fitness; Losers; Mitochondrial dysregulation; Proteostasis; Winners

DOI:  https://doi.org/10.1016/j.ceb.2024.102323