bims-proteo 2023-04-23 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒04‒23
28 papers selected by
Eric Chevet
INSERM

Ubiquitin-binding autophagic receptors in yeast: Cue5 and beyond.
Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function.
Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.
Nuclear and cytoplasmic spatial protein quality control is coordinated by nuclear-vacuolar junctions and perinuclear ESCRT.
A TRAF-like E3 ubiquitin ligase TrafE coordinates ESCRT and autophagy in endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.
Hsp90 provides a platform for kinase dephosphorylation by PP5.
Alzheimer's disease-associated mutant ubiquitin (UBB+1) is secreted through an autophagosome-like vesicle-mediated unconventional pathway.
Identification of LMAN1 and SURF4 dependent secretory cargoes.
The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.
The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts.
Insulin secretion deficits in a Prader-Willi syndrome β-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.
A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila.
The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains.
Proteomic discovery of chemical probes that perturb protein complexes in human cells.
High-resolution structure of mammalian PI31â€"20S proteasome complex reveals mechanism of proteasome inhibition.
GCN2 kinase-mediated upregulation of ubiquitin C maintains intracellular glutamine level and tRNAGln (CUG) charging under amino acid starvation.
Co-targeting BCL-XL and BCL-2 by PROTAC 753B eliminates leukemia cells and enhances efficacy of chemotherapy by targeting senescent cells.
APP deficiency and HTRA2 modulates PrPc proteostasis in human cancer cells.
A systematic proximity ligation approach to studying protein-substrate specificity identifies the substrate spectrum of the Ssh1 translocon.
Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice.
NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.
Structural inventory of cotranslational protein folding by the eukaryotic RAC complex.
A novel method for visualizing in-vivo rates of protein degradation provides insight into how TRIM28 regulates muscle size.
Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.
Lenalidomide Stabilizes Protein-Protein Complexes by Turning Labile Intermolecular H-Bonds into Robust Interactions.
A new avenue for molecular glues: Rapid discovery of a NFKB1 degrader.
A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression.
The proteomic landscape of genome-wide genetic perturbations.

Autophagy. 2023 Apr 16. 1-5

Ubiquitin-binding autophagic receptors in yeast: Cue5 and beyond.

Theresah Nana Ama Mensah, Ankit Shroff, Taras Y Nazarko.

  The selectivity in selective macroautophagy/autophagy pathways is achieved via selective autophagy receptors (SARs) - proteins that bind a ligand on the substrate to be degraded and an Atg8-family protein on the growing autophagic membrane, phagophore, effectively bridging them. In mammals, the most common ligand of SARs is ubiquitin, a small protein modifier that tags substrates for their preferential degradation by autophagy. Consequently, most common SARs are ubiquitin-binding SARs, such as SQSTM1/p62 (sequestosome 1). Surprisingly, there is only one SAR of this type in yeast - Cue5, which acts as the receptor for aggrephagy and proteaphagy - pathways that remove ubiquitinated protein aggregates and proteasomes, respectively. However, recent studies described ubiquitin-dependent autophagic pathways that do not require Cue5, e.g. the stationary phase lipophagy for lipid droplets or nitrogen starvation-induced mitophagy for mitochondria. What is the role of ubiquitin in these pathways? Here, we propose that ubiquitinated lipid droplets and mitochondria are recognized by alternative ubiquitin-binding SARs. Our analysis identifies proteins that could potentially fulfill this role in yeast. We think that matching of ubiquitin-dependent (but Cue5-independent) autophagic pathways with ubiquitin- and Atg8-binding proteins enlisted here might uncover novel ubiquitin-binding SARs in yeast.Abbreviations: AIM: Atg8-family interacting motif; CUE: coupling of ubiquitin conjugation to ER degradation; ERMES: endoplasmic reticulum-mitochondria encounter structure; HECT: homologous to the E6-AP carboxyl terminus; LD: lipid droplet; SAR: selective autophagy receptor; SGD: Saccharomyces Genome Database; UBA: ubiquitin-associated; UBX: ubiquitin regulatory X; UIM: ubiquitin-interacting motif.

Keywords:  Cue5; autophagic receptor; selective autophagy; selective autophagy receptor; ubiquitin-binding protein; ubiquitin-binding receptor

DOI:  https://doi.org/10.1080/15548627.2023.2196878
bioRxiv. 2023 Apr 03. pii: 2023.04.03.535444. [Epub ahead of print]

Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function.

Brian G Peterson, Jiwon Hwang, Jennifer E Russ, Jeremy Schroeder, Peter L Freddolino, Ryan D Baldridge.

Misfolded endoplasmic reticulum proteins are degraded through a process called endoplasmic reticulum associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identified several regions required for different Hrd1 functions. Most surprisingly, we found two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we defined roles for disordered regions between structural elements that were required for Hrd1â€™s ability to autoubiquitinate and interact with substrate. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the endoplasmic reticulum membrane.

DOI: https://doi.org/10.1101/2023.04.03.535444
Nat Commun. 2023 04 15. 14(1): 2160

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

Leo Kiss, Tyler Rhinesmith, Jakub Luptak, Claire F Dickson, Jonas Weidenhausen, Shannon Smyly, Ji-Chun Yang, Sarah L Maslen, Irmgard Sinning, David Neuhaus, Dean Clift, Leo C James.

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.

DOI: https://doi.org/10.1038/s41467-023-37504-x
Nat Cell Biol. 2023 Apr 20.

Nuclear and cytoplasmic spatial protein quality control is coordinated by nuclear-vacuolar junctions and perinuclear ESCRT.

Emily M Sontag, Fabián Morales-Polanco, Jian-Hua Chen, Gerry McDermott, Patrick T Dolan, Daniel Gestaut, Mark A Le Gros, Carolyn Larabell, Judith Frydman.

Effective protein quality control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we reveal the coordination of nuclear and cytoplasmic spatial PQC. Cytoplasmic misfolded proteins concentrate in a cytoplasmic juxtanuclear quality control compartment, while nuclear misfolded proteins sequester into an intranuclear quality control compartment (INQ). Particle tracking reveals that INQ and the juxtanuclear quality control compartment converge to face each other across the nuclear envelope at a site proximal to the nuclear-vacuolar junction marked by perinuclear ESCRT-II/III protein Chm7. Strikingly, convergence at nuclear-vacuolar junction contacts facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. Finding that nuclear-vacuolar contact sites are cellular hubs of spatial PQC to facilitate vacuolar clearance of nuclear and cytoplasmic inclusions highlights the role of cellular architecture in proteostasis maintenance.

DOI: https://doi.org/10.1038/s41556-023-01128-6
Elife. 2023 Apr 18. pii: e85727. [Epub ahead of print]12

A TRAF-like E3 ubiquitin ligase TrafE coordinates ESCRT and autophagy in endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.

Lyudmil Raykov, Manon Mottet, Jahn Nitschke, Thierry Soldati.

  Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. This severe stress is recognised and controlled by the endosomal sorting complex required for transport (ESCRT) and the autophagy machineries, which are recruited to damaged membranes to either repair or to remove membrane remnants. Yet, insight is limited about how damage is sensed and which effectors lead to extensive tagging of the damaged organelles with signals, such as K63-polyubiquitin, required for the recruitment of membrane repair or removal machineries. To explore the key factors responsible for detection and marking of damaged compartments, we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase, TrafE, that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical compounds. TrafE acts at the intersection of ESCRT and autophagy pathways and plays a key role in functional recruitment of the ESCRT subunits ALIX, Vps32 and Vps4 to damage sites. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of mycobacteria as well as ESCRT-mediated and autophagy-mediated endolysosomal membrane damage repair, resulting in early cell death.

Keywords:  cell biology; dictyostelium; infectious disease; microbiology

DOI:  https://doi.org/10.7554/eLife.85727
Nat Commun. 2023 04 17. 14(1): 2197

Hsp90 provides a platform for kinase dephosphorylation by PP5.

Maru Jaime-Garza, Carlos A Nowotny, Daniel Coutandin, Feng Wang, Mariano Tabios, David A Agard.

The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf and Cdc37 in an Hsp90-dependent manner. Although dephosphorylating Cdc37 has been proposed as a mechanism for releasing Hsp90-bound kinases, here we show that Hsp90 bound kinases sterically inhibit Cdc37 dephosphorylation indicating kinase release must occur before Cdc37 dephosphorylation. Our cryo-EM structure of PP5 in complex with Hsp90:Cdc37:CRaf reveals how Hsp90 both activates PP5 and scaffolds its association with the bound CRaf to dephosphorylate phosphorylation sites neighboring the kinase domain. Thus, we directly show how Hsp90's role in maintaining protein homeostasis goes beyond folding and activation to include post translationally modifying its client kinases.

DOI: https://doi.org/10.1038/s41467-023-37659-7
Biochim Biophys Acta Gene Regul Mech. 2023 Apr 17. pii: S1874-9399(23)00031-7. [Epub ahead of print] 194936

Alzheimer's disease-associated mutant ubiquitin (UBB+1) is secreted through an autophagosome-like vesicle-mediated unconventional pathway.

Ajay R Wagh, Prasad Sulakshane, Michael H Glickman.

  Misfolded protein aggregation at both intracellular and extracellular milieus is thought to be the major etiology of Alzheimer's disease (AD). UBB+1, a frameshift variant of the ubiquitin B gene (UBB) results in a folded ubiquitin domain fused to a flexible unstructured extension. Accumulation of UBB+1 in extracellular plaques in the brains of AD patients undoubtedly suggests a role of the ubiquitin-proteasome system in AD. However, the exact mechanism of extracellular secretion of UBB+1 remains unknown. In an attempt to understand the molecular mechanism of UBB+1 secretion, we performed a survey of secretory pathways and identified the involvement of unconventional autophagosome-mediated UBB+1 secretion. Expression of UBB+1 was sufficient to stimulate LC3B/Atg8 conversion from LC3B-I to LC3B-II, which indicates initiation of the autophagy pathway. Furthermore, deficiency of ATG5 - a key player in autophagosome formation - inhibited UBB+1 secretion. Based on immunofluorescence 3D structured illumination (SIM) microscopy and co-immunoprecipitation, we provide evidence that UBB+1 is associated with the secretory autophagosome marker, SEC22B, while HSP90 possibly acts as a carrier. Using LC-MS/MS and mutagenesis we found that in cells, UBB+1 is ubiquitinated on lysine 11, 29, and 48, however, this ubiquitination does not contribute to its secretion. By contrast, proteasome or lysosome inhibition slightly enhanced secretion. Taken together, this study suggests that by ridding cells of UBB+1, secretory autophagosomes may alleviate the cellular stress associated with UBB+1, yet simultaneously mediate the spreading of a mutant specie with disordered characteristics to the extracellular milieu.

Keywords:  Autophagosome mediated secretion; Autophagy; HSP90; Lysosome; Mutant ubiquitin; Proteasome; SEC22B; UBB(+1); Ubiquitination

DOI:  https://doi.org/10.1016/j.bbagrm.2023.194936
bioRxiv. 2023 Apr 06. pii: 2023.04.06.535922. [Epub ahead of print]

Identification of LMAN1 and SURF4 dependent secretory cargoes.

Vi T Tang, Prabhodh S Abbineni, Felipe da Veiga Leprevost, Venkatesha Basrur, Brian T Emmer, Alexey I Nesvizhskii, David Ginsburg.

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles/tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif.

DOI: https://doi.org/10.1101/2023.04.06.535922
J Biol Chem. 2023 Apr 13. pii: S0021-9258(23)01740-4. [Epub ahead of print] 104712

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.

Henning Arlt, Babu Raman, Yasmina Filali-Mouncef, Yan Hu, Alexandre Leytens, Ralph Hardenberg, Rodrigo Guimarães, Franziska Kriegenburg, Muriel Mari, Iwona I Smaczynska-de Rooij, Kathryn R Ayscough, Jörn Dengjel, Christian Ungermann, Fulvio Reggiori.

  Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.

Keywords:  Atg9; Autophagy; PAS; SNX; Vps1; autophagosome; dynamin; phagophore; retromer; sorting nexin

DOI:  https://doi.org/10.1016/j.jbc.2023.104712
Elife. 2023 Apr 18. pii: e86030. [Epub ahead of print]12

The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts.

Han Nim Lee, Jenu Chacko, Ariadna Gonzalez Solís, Kuo-En Chen, Jessica A S Barros, Santiago Signorelli, A Harvey Millar, Richard David Vierstra, Kevin W Eliceiri, Marisa S Otegui.

  The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.

Keywords:  A. thaliana; cell biology; plant biology

DOI:  https://doi.org/10.7554/eLife.86030
PLoS Genet. 2023 Apr 17. 19(4): e1010710

Insulin secretion deficits in a Prader-Willi syndrome β-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.

Erik A Koppes, Marie A Johnson, James J Moresco, Patrizia Luppi, Dale W Lewis, Donna B Stolz, Jolene K Diedrich, John R Yates, Ronald C Wek, Simon C Watkins, Susanne M Gollin, Hyun Jung Park, Peter Drain, Robert D Nicholls.

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in β-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS β-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS β-cells. Consistent with reduced ER chaperones levels, PWS INS-1 β-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS β-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic β-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and β-cell secretory pathway function.

DOI: https://doi.org/10.1371/journal.pgen.1010710
Cell Mol Life Sci. 2023 Apr 22. 80(5): 129

A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila.

Huifang Li, Zhenghong Yu, Zikang Niu, Yun Cheng, Zhenhao Wei, Yafei Cai, Fei Ma, Lanxin Hu, Jiejie Zhu, Wei Zhang.

  Ufmylation is a recently identified small ubiquitin-like modification, whose biological function and relevant cellular targets are poorly understood. Here we present evidence of a neuroprotective role for Ufmylation involving Autophagy-related gene 9 (Atg9) during Drosophila aging. The Ufm1 system ensures the health of aged neurons via Atg9 by coordinating autophagy and mTORC1, and maintaining mitochondrial homeostasis and JNK (c-Jun N-terminal kinase) activity. Neuron-specific expression of Atg9 suppresses the age-associated movement defect and lethality caused by loss of Ufmylation. Furthermore, Atg9 is identified as a conserved target of Ufm1 conjugation mediated by Ddrgk1, a critical regulator of Ufmylation. Mammalian Ddrgk1 was shown to be indispensable for the stability of endogenous Atg9A protein in mouse embryonic fibroblast (MEF) cells. Taken together, our findings might have important implications for neurodegenerative diseases in mammals.

Keywords:  Life span; Oxidative stress; Patj; Puc; Uba5; Ufl1

DOI:  https://doi.org/10.1007/s00018-023-04778-9
PLoS Biol. 2023 Apr;21(4): e3001995

The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains.

Alexandra G Knorr, Timur Mackens-Kiani, Joanna Musial, Otto Berninghausen, Thomas Becker, Birgitta Beatrix, Roland Beckmann.

Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tunnel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together suggesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel.

DOI: https://doi.org/10.1371/journal.pbio.3001995
Mol Cell. 2023 Apr 18. pii: S1097-2765(23)00239-3. [Epub ahead of print]

Proteomic discovery of chemical probes that perturb protein complexes in human cells.

Michael R Lazear, Jarrett R Remsberg, Martin G Jaeger, Katherine Rothamel, Hsuan-Lin Her, Kristen E DeMeester, Evert Njomen, Simon J Hogg, Jahan Rahman, Landon R Whitby, Sang Joon Won, Michael A Schafroth, Daisuke Ogasawara, Minoru Yokoyama, Garrett L Lindsey, Haoxin Li, Jason Germain, Sabrina Barbas, Joan Vaughan, Thomas W Hanigan, Vincent F Vartabedian, Christopher J Reinhardt, Melissa M Dix, Seong Joo Koo, Inha Heo, John R Teijaro, Gabriel M Simon, Brahma Ghosh, Omar Abdel-Wahab, Kay Ahn, Alan Saghatelian, Bruno Melillo, Stuart L Schreiber, Gene W Yeo, Benjamin F Cravatt.

  Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

Keywords:  activity-based protein profiling; chemical probe; covalent; cysteine; proteasome; protein complexes; proteomics; size-exclusion chromatography; spliceosome

DOI:  https://doi.org/10.1016/j.molcel.2023.03.026
bioRxiv. 2023 Apr 07. pii: 2023.04.03.535455. [Epub ahead of print]

High-resolution structure of mammalian PI31â€"20S proteasome complex reveals mechanism of proteasome inhibition.

Hao-Chi Hsu, Jason Wang, Abbey Kjellgren, Huilin Li, George N DeMartino.

Proteasome-catalyzed protein degradation mediates and regulates critical aspects of many cellular functions and is an important element of proteostasis in health and disease. Proteasome function is determined in part by the types of proteasome holoenzymes formed between the 20S core particle that catalyzes peptide bond hydrolysis and any of multiple regulatory proteins to which it binds. One of these regulators, PI31, was previously identified as an in vitro 20S proteasome inhibitor, but neither the molecular mechanism nor the possible physiologic significance of PI31-mediated proteasome inhibition has been clear. Here we report a high- resolution cryo-EM structure of the mammalian 20S proteasome in complex with PI31. The structure shows that two copies of the intrinsically-disordered carboxyl-terminus of PI31 are present in the central cavity of the closed-gate conformation of the proteasome and interact with proteasome catalytic sites in a manner that blocks proteolysis of substrates but resists their own degradation. The two inhibitory polypeptide chains appear to originate from PI31 monomers that enter the catalytic chamber from opposite ends of the 20S cylinder. We present evidence that PI31 can inhibit proteasome activity in mammalian cells and may serve regulatory functions for the control of cellular proteostasis.

DOI: https://doi.org/10.1101/2023.04.03.535455
FEBS Lett. 2023 Apr 20.

GCN2 kinase-mediated upregulation of ubiquitin C maintains intracellular glutamine level and tRNAGln (CUG) charging under amino acid starvation.

Yusuke Tsukamoto, Yumi Nakamura, Makoto Hirata, Daisuke Okuzaki, Ryuichi Sakate, Tomonori Kimura.

Each tRNA is aminoacylated (charged) with a genetic codon-specific amino acid. It remains unclear what factors are associated with tRNA charging and how tRNA charging is maintained. By using the individual tRNA acylation PCR (i-tRAP) method, we found that the charging ratio of tRNAGln (CUG) reflects cellular glutamine level. When uncharged tRNAGln (CUG) increased under amino acid starvation, the kinase GCN2, which is a key stimulator of the integrated stress response, was activated. Activation of GCN2 led to the upregulation of ubiquitin C (UBC) expression. Upregulated UBC, in turn, suppressed the further reduction of tRNAGln (CUG) charging levels. Thus, tRNA charging is sensitive to intracellular nutrient status and is an important initiator of intracellular signaling.

DOI: https://doi.org/10.1002/1873-3468.14628
Haematologica. 2023 Apr 20.

Co-targeting BCL-XL and BCL-2 by PROTAC 753B eliminates leukemia cells and enhances efficacy of chemotherapy by targeting senescent cells.

Yannan Jia, Lina Han, Cassandra L Ramage, Zhe Wang, Connie C Weng, Lei Yang, Simona Colla, Helen Ma, Weiguo Zhang, Michael Andreeff, Naval Daver, Nitin Jain, Naveen Pemmaraju, Kapil Bhalla, Satu Mustjoki, Peiyi Zhang, Guangrong Zheng, Daohong Zhou, Qi Zhang, Marina Konopleva.

BCL-XL and BCL-2 are key anti-apoptotic proteins and validated cancer targets. 753B is a novel BCL-XL/BCL-2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-XL/BCL-2 ubiquitination and degradation selectively in cells expressing VHL. Because platelets lack VHL expression, 753B spares on-target platelet toxicity caused by the first generation dual BCL-XL/BCL-2 inhibitor navitoclax (ABT-263). Here, we report pre-clinical single-agent activity of 753B against different leukemia subsets. 753B effectively reduced cell viability and induced dose-dependent degradation of BCL-XL and BCL-2 in a subset of hematopoietic cell lines, AML primary samples and in vivo PDX AML model. We further demonstrated the senolytic activity of 753B which enhanced the efficacy of chemotherapy by targeting chemotherapy-induced cellular senescence. These results provide a pre-clinical rationale for the utility of 753B in AML therapy, and suggest that 753B could produce an added therapeutic benefit by overcoming cellular senescence-induced chemoresistance when combined with chemotherapy.

DOI: https://doi.org/10.3324/haematol.2022.281915
BBA Adv. 2022 ;2 100035

APP deficiency and HTRA2 modulates PrPc proteostasis in human cancer cells.

Denis S F Biard, Rafika Jarray, Nicolas Rebergue, François Leteurtre, Dulce Papy-Garcia.

  Cellular protein homeostasis (proteostasis) requires an accurate balance between protein biosynthesis, folding, and degradation, and its instability is causally related to human diseases and cancers. Here, we created numerous engineered cancer cell lines targeting APP (amyloid ß precursor protein) and/or PRNP (cellular prion) genes and we showed that APP knocking-down impaired PRNP mRNA level and vice versa, suggesting a link between their gene regulation. PRNPKD, APPKD and PRNPKD/APPKD HeLa cells encountered major difficulties to grow in a 3D tissue-like environment. Unexpectedly, we found a cytoplasmic accumulation of the PrPc protein without PRNP gene up regulation, in both APPKD and APPKO HeLa cells. Interestingly, APP and/or PRNP gene ablation enhanced the chaperone/serine protease HTRA2 gene expression, which is a protein processing quality factor involved in Alzheimer's disease. Importantly, HTRA2 gene silencing decreased PRNP mRNA level and lowered PrPc protein amounts, and conversely, HTRA2 overexpression increased PRNP gene regulation and enhanced membrane-anchored and cytoplasmic PrPc fractions. PrPc, APP and HTRA2 destabilized membrane-associated CD24 protein, suggesting changes in the lipid raft structure. Our data show for the first time that APP and the dual chaperone/serine protease HTRA2 protein could modulate PrPc proteostasis hampering cancer cell behavior.

Keywords:  APP; CD24; CRISPR-Cas9; HTRA2; PRNP; PrPc; RNA interference; cancer cells; pEBVsiRNA; proteostasis

DOI:  https://doi.org/10.1016/j.bbadva.2021.100035
EMBO J. 2023 Apr 19. e113385

A systematic proximity ligation approach to studying protein-substrate specificity identifies the substrate spectrum of the Ssh1 translocon.

Nir Cohen, Naama Aviram, Maya Schuldiner.

  Many cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein-protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins - substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel-lctiv (CELlular biotin-Ligation for Capturing Transient Interactions in vivo), uses high-throughput pairwise proximity biotin ligation for comparing PPIs systematically and in vivo. As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel-lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel-lctiv can provide direct information on substrate specificity even for highly homologous proteins.

Keywords:  BirA; Sec61; Ssh1; proximity labeling; translocation

DOI:  https://doi.org/10.15252/embj.2022113385
Nat Microbiol. 2023 Apr 20.

Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice.

Yanyan Zheng, Liqiu Wang, Qingxiang Liu, Huifang Xian, Chenqiu Zhang, Sihui Cai, Shuai Yang, Shouheng Jin, Jun Cui.

Microglia and astrocytes are subgroups of brain glia cells that support and protect neurons within the central nervous system (CNS). At early stages of viral infection in the CNS, they are predominant responding cells and lead to recruitment of peripheral immune cells for viral clearance. Inhibitor of nuclear factor κB kinase subunit epsilon (IKKi) is critical for type I interferon signalling and inflammation, which modulate heterogenic immune responses during CNS infection. Balanced autophagy is vital to maintain brain integrity, yet regulation of autophagy and immune activity within brain glia cells is poorly understood. Here we identify SHISA9 as an autophagy cargo receptor that mediates the autophagy-dependent degradation of IKKi during herpes simplex virus type 1 infection. IKKi is recognized by SHISA9 through unanchored K48-linked poly-ubiquitin chains and bridged to autophagosome membrane components GABARAPL1. Single-cell RNA sequencing analysis shows that SHISA9 has temporal characteristics while modulating both antiviral and inflammatory responses in microglia and astrocytes at different stages during viral infection. We found that Shisa9-/- mice are highly susceptible to herpes simplex virus encephalitis, have pathogenic astrocytes and display more severe neuroinflammation compared with wild-type mice. Taken together, our study unravels a critical role of selective autophagy by orchestrating immune heterogeneity of different CNS resident cells through the SHISA9-IKKi axis.

DOI: https://doi.org/10.1038/s41564-023-01357-3
Cell Rep. 2023 Apr 20. pii: S2211-1247(23)00383-2. [Epub ahead of print]42(5): 112372

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Congxin Sun, Elena Seranova, Malkiel A Cohen, Miruna Chipara, Jennie Roberts, Dewi Astuti, Adina M Palhegyi, Animesh Acharjee, Lucia Sedlackova, Tetsushi Kataura, Elsje G Otten, Prashanta K Panda, Samuel Lara-Reyna, Miriam E Korsgen, Kevin J Kauffman, Alejandro Huerta-Uribe, Malgorzata Zatyka, Luiz F S E Silva, Jorge Torresi, Shupei Zhang, Georgina W Hughes, Carl Ward, Erich R Kuechler, David Cartwright, Sergey Trushin, Eugenia Trushina, Gaurav Sahay, Yosef Buganim, Gareth G Lavery, Joerg Gsponer, Daniel G Anderson, Eva-Maria Frickel, Tatiana R Rosenstock, Timothy Barrett, Oliver D K Maddocks, Daniel A Tennant, Haoyi Wang, Rudolf Jaenisch, Viktor I Korolchuk, Sovan Sarkar.

  Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Keywords:  CP: Cell biology; CP: Metabolism; NAD; NADases; NAM; NMN; NR; autophagy; cell death; cell survival, human embryonic stem cell-derived neurons; mitochondria; nicotinamide; nicotinamide adenine dinucleotide; nicotinamide mononucleotide; nicotinamide riboside

DOI:  https://doi.org/10.1016/j.celrep.2023.112372
Nat Struct Mol Biol. 2023 Apr 20.

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex.

Miglė Kišonaitė, Klemens Wild, Karine Lapouge, Genís Valentín Gesé, Nikola Kellner, Ed Hurt, Irmgard Sinning.

The challenge of nascent chain folding at the ribosome is met by the conserved ribosome-associated complex (RAC), which forms a chaperone triad with the Hsp70 protein Ssb in fungi, and consists of the non-canonical Hsp70 Ssz1 and the J domain protein Zuotin (Zuo1). Here we determine cryo-EM structures of Chaetomium thermophilum RAC bound to 80S ribosomes. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. It is held together by a tight interaction between the Ssz1 substrate-binding domain and the Zuo1 N terminus, and additional contacts between the Ssz1 nucleotide-binding domain and Zuo1 J- and Zuo1 homology domains, which form a rigid unit. The Zuo1 HPD motif conserved in J-proteins is masked in a non-canonical interaction by the Ssz1 nucleotide-binding domain, and allows the positioning of Ssb for activation by Zuo1. Overall, we provide the basis for understanding how RAC cooperates with Ssb in a dynamic nascent chain interaction and protein folding.

DOI: https://doi.org/10.1038/s41594-023-00973-1
iScience. 2023 Apr 21. 26(4): 106526

A novel method for visualizing in-vivo rates of protein degradation provides insight into how TRIM28 regulates muscle size.

Nathaniel D Steinert, Kent W Jorgenson, Kuan-Hung Lin, Jake B Hermanson, Jake L Lemens, Troy A Hornberger.

  Skeletal muscle size is controlled by the balance between protein synthesis and protein degradation. Given the essential role of skeletal muscle in maintaining a high quality of life, understanding the mechanisms that modulate this balance are of critical importance. Previously, we demonstrated that muscle-specific knockout of TRIM28 reduces muscle size and function and in the current study, we discovered that this effect is associated with an increase in protein degradation and a dramatic reduction in the expression of Mettl21c. Importantly, we also determined that overexpression of Mettl21c is sufficient to induce hypertrophy in both control and TRIM28 knockout muscles. Moreover, we developed a simple pulse-chase biorthogonal non-canonical amino acid tagging technique that enabled us to visualize the in vivo rate of protein degradation, and with this technique were able to conclude that the hypertrophic effect of Mettl21c is due, at least in part, to an inhibition of protein degradation.

Keywords:  Biochemistry; Cell biology; Developmental biology

DOI:  https://doi.org/10.1016/j.isci.2023.106526
STAR Protoc. 2023 Apr 18. pii: S2666-1667(23)00194-6. [Epub ahead of print]4(2): 102236

Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.

Keisuke Tabata, Marika Saeki, Tamotsu Yoshimori, Maho Hamasaki.

  Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes. For complete details on the use and execution of this protocol, please refer to Teranishi et al. (2022).1.

Keywords:  Cell Biology; Cell Culture; High-throughput Screening; Microscopy

DOI:  https://doi.org/10.1016/j.xpro.2023.102236
J Med Chem. 2023 Apr 21.

Lenalidomide Stabilizes Protein-Protein Complexes by Turning Labile Intermolecular H-Bonds into Robust Interactions.

Marina Miñarro-Lleonar, Andrea Bertran-Mostazo, Jorge Duro, Xavier Barril, Jordi Juárez-Jiménez.

Targeted protein degradation is a promising therapeutic strategy, spearheaded by the anti-myeloma drugs lenalidomide and pomalidomide. These drugs stabilize very efficiently the complex between the E3 ligase Cereblon (CRBN) and several non-native client proteins (neo-substrates), including the transcription factors Ikaros and Aiolos and the enzyme Caseine Kinase 1α (CK1α,), resulting in their degradation. Although the structures for these complexes have been determined, there are no evident interactions that can account for the high efficiency of formation of the ternary complex. We show that lenalidomide's stabilization of the CRBN-CK1α complex is largely due to hydrophobic shielding of intermolecular hydrogen bonds. We also find a quantitative relationship between hydrogen bond robustness and binding affinities of the ternary complexes. These results pave the way to further understand cooperativity effects in drug-induced protein-protein complexes and could help in the design of improved molecular glues and more efficient protein degraders.

DOI: https://doi.org/10.1021/acs.jmedchem.2c01692
Cell Chem Biol. 2023 Apr 20. pii: S2451-9456(23)00088-0. [Epub ahead of print]30(4): 340-342

A new avenue for molecular glues: Rapid discovery of a NFKB1 degrader.

Angela T Fan, Bridget T Boyle, Fleur M Ferguson.

Targeted protein degradation using molecular glues is a powerful method for targeting traditionally undruggable proteins. One challenge in molecular glue discovery is the absence of rational discovery methods. Here, King et al. leverage covalent library screening with chemoproteomics platforms to rapidly discover a molecular glue targeting NFKB1 via UBE2D recruitment.

DOI: https://doi.org/10.1016/j.chembiol.2023.04.002
PLoS Comput Biol. 2023 Apr 17. 19(4): e1010995

A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression.

Salim Ghannoum, Damiano Fantini, Muhammad Zahoor, Veronika Reiterer, Santosh Phuyal, Waldir Leoncio Netto, Øystein Sørensen, Arvind Iyer, Debarka Sengupta, Lina Prasmickaite, Gunhild Mari Mælandsmo, Alvaro Köhn-Luque, Hesso Farhan.

Our understanding of how speed and persistence of cell migration affects the growth rate and size of tumors remains incomplete. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation, a phenomenon often linked to alterations of cell migration. Golgi fragmentation was induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the experimentally obtained migration and invasion traits of Giantin depleted breast cancer cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

DOI: https://doi.org/10.1371/journal.pcbi.1010995
Cell. 2023 Apr 17. pii: S0092-8674(23)00300-8. [Epub ahead of print]

The proteomic landscape of genome-wide genetic perturbations.

Christoph B Messner, Vadim Demichev, Julia Muenzner, Simran K Aulakh, Natalie Barthel, Annika Röhl, Lucía Herrera-Domínguez, Anna-Sophia Egger, Stephan Kamrad, Jing Hou, Guihong Tan, Oliver Lemke, Enrica Calvani, Lukasz Szyrwiel, Michael Mülleder, Kathryn S Lilley, Charles Boone, Georg Kustatscher, Markus Ralser.

  Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Keywords:  Saccharomyces cerevisiae; data-independent acquisition; deletion; functional genomics; functional proteomics; gene annotation; high throughput; knockout; quantitative proteomics; systems biology

DOI:  https://doi.org/10.1016/j.cell.2023.03.026