bims-proteo 2023-08-20 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒08‒20
29 papers selected by
Eric Chevet
INSERM

Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control.
Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation.
Identification of KLHDC2 as an efficient proximity-induced degrader of K-RAS, STK33, β-catenin, and FoxP3.
The ubiquitin-like modifier FAT10 covalently modifies HUWE1 and strengthens the interaction of AMBRA1 and HUWE1.
C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways.
The atlastin ER morphogenic proteins promote formation of a membrane penetration site during non-enveloped virus entry.
MAVS-loaded unanchored Lys63-linked polyubiquitin chains activate the RIG-I-MAVS signaling cascade.
From Thalidomide to Rational Molecular Glue Design for Targeted Protein Degradation.
E4 ubiquitin ligase promotes mitofusin turnover and mitochondrial stress response.
A chemical probe unravels the reactive proteome of health-associated catechols.
Molecular mechanism for activation of the 26S proteasome by ZFAND5.
Control of mRNA fate by its encoded nascent polypeptide.
Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal.
Post-translational modifications of ATG8 proteins - an emerging mechanism of autophagy control.
Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired.
Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation.
Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity.
Opportunities and challenges of protein-based targeted protein degradation.
An interaction between OTULIN and SCRIB uncovers roles for linear ubiquitination in planar cell polarity.
The NRF2-p97-NRF2 negative feedback loop.
Selective inhibition of hsp90 paralogs: Structure and binding studies uncover the role of helix 1 in Grp94-selective ligand binding.
Parallel proteomics and phosphoproteomics defines starvation signal specific processes in cell quiescence.
Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import.
Development of an OTUD1 ubiquitin variant inhibitor.
Regulation of translation by ribosomal RNA pseudouridylation.
Gelation of cytoplasmic expanded CAG RNA repeats suppresses global protein synthesis.
Protein interaction network analysis of mTOR signaling reveals modular organization.
Autophagy in intestinal epithelial cells prevents gut inflammation.
Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum.

Sci Adv. 2023 Aug 18. 9(33): eadh3635

Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control.

Ryosuke Ishimura, Sota Ito, Gaoxin Mao, Satoko Komatsu-Hirota, Toshifumi Inada, Nobuo N Noda, Masaaki Komatsu.

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and, subsequently, CDK5RAP3, an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation, the E3 complex associated with ufmylated RPL26 on the 60S subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results provide insights into not only the molecular basis of the ufmylation but also its role in proteostasis.

DOI: https://doi.org/10.1126/sciadv.adh3635
bioRxiv. 2023 Aug 03. pii: 2023.08.01.551382. [Epub ahead of print]

Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation.

Heejun Choi, Ya-Cheng Liao, Young J Yoon, Jonathan Grimm, Luke D Lavis, Robert H Singer, Jennifer Lippincott-Schwartz.

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.

DOI: https://doi.org/10.1101/2023.08.01.551382
Cell Chem Biol. 2023 Aug 09. pii: S2451-9456(23)00233-7. [Epub ahead of print]

Identification of KLHDC2 as an efficient proximity-induced degrader of K-RAS, STK33, β-catenin, and FoxP3.

Sascha Röth, Nur Mehpare Kocaturk, Preethi S Sathyamurthi, Bill Carton, Matthew Watt, Thomas J Macartney, Kwok-Ho Chan, Albert Isidro-Llobet, Agnieszka Konopacka, Markus A Queisser, Gopal P Sapkota.

  Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, β-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells.

Keywords:  E3 ligase; FoxP3; KLHDC2; PROTAC; RAS; STK33; TPD; cancer; degron; β-catenin

DOI:  https://doi.org/10.1016/j.chembiol.2023.07.006
PLoS One. 2023 ;18(8): e0290002

The ubiquitin-like modifier FAT10 covalently modifies HUWE1 and strengthens the interaction of AMBRA1 and HUWE1.

Stefanie Mueller, Johanna Bialas, Stella Ryu, Nicola Catone, Annette Aichem.

The ubiquitin-like modifier FAT10 is highly upregulated under inflammatory conditions and targets its conjugation substrates to the degradation by the 26S proteasome. This process termed FAT10ylation is mediated by an enzymatic cascade and includes the E1 activating enzyme ubiquitin-like modifier activating enzyme 6 (UBA6), the E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1) and E3 ligases, such as Parkin. In this study, the function of the HECT-type ubiquitin E3 ligase HUWE1 was investigated as a putative E3 ligase and/or conjugation substrate of FAT10. Our data provide strong evidence that HUWE1 is FAT10ylated in a UBA6 and FAT10 diglycine-dependent manner in vitro and in cellulo and that the HUWE1-FAT10 conjugate is targeted to proteasomal degradation. Since the mutation of all relevant cysteine residues within the HUWE1 HECT domain did not abolish FAT10 conjugation, a role of HUWE1 as E3 ligase for FAT10ylation is rather unlikely. Moreover, we have identified the autophagy-related protein AMBRA1 as a new FAT10 interaction partner. We show that the HUWE1-FAT10 conjugate formation is diminished in presence of AMBRA1, while the interaction between AMBRA1 and HUWE1 is strengthened in presence of FAT10. This implies a putative interplay of all three proteins in cellular processes such as mitophagy.

DOI: https://doi.org/10.1371/journal.pone.0290002
J Biol Chem. 2023 Aug 16. pii: S0021-9258(23)02194-4. [Epub ahead of print] 105166

C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways.

Sophia Hasenjäger, Andrea Bologna, Lars-Oliver Essen, Roberta Spadaccini, Christof Taxis.

  Protein quality control (PQC) mechanisms are essential for degradation of misfolded or dysfunctional proteins. An essential part of protein homeostasis is recognition of defective proteins by PQC components and their elimination by the ubiquitin-proteasome system, often concentrating on protein termini as indicators of protein integrity. Changes in amino acid composition of C-terminal ends arise through protein disintegration, alternative splicing or during the translation step of protein synthesis from premature termination or translational stop-codon read-through. We characterized reporter protein stability using light-controlled exposure of random C-terminal peptides (CtPC) in budding yeast revealing stabilizing and destabilizing features of amino acids at positions -5 to -1 of the C-terminus. The (de)stabilization properties of CtPC-degrons depend on amino acid identity, position as well as composition of the C-terminal sequence and are transferable. Evolutionary pressure towards stable proteins in yeast is evidenced by amino acid residues under-represented in cytosolic and nuclear proteins at corresponding C-terminal positions, but over-represented in unstable CtPC-degrons, and vice versa. Furthermore, analysis of translational stop-codon read-through peptides suggested that such extended proteins have destabilizing C-termini. PQC pathways targeting CtPC-degrons involved the ubiquitin-protein ligase Doa10 and the cullin-RING E3 ligase (CRL) SCFDas1. Overall, our data suggest a proteome protection mechanism that targets proteins with unnatural C-termini by recognizing a surprisingly large number of C-terminal sequence variants.

Keywords:  C-terminus; optogenetics; proteasome; protein degradation; protein quality control; proteolysis; ubiquitin-proteasome system

DOI:  https://doi.org/10.1016/j.jbc.2023.105166
J Virol. 2023 Aug 14. e0075623

The atlastin ER morphogenic proteins promote formation of a membrane penetration site during non-enveloped virus entry.

Madison Pletan, Xiaofang Liu, Grace Cha, Yu-Jie Chen, Jeffrey Knupp, Billy Tsai.

  During entry, non-enveloped viruses penetrate a host membrane to cause infection, although how this is accomplished remains enigmatic. Polyomaviruses (PyVs) are non-enveloped DNA viruses that penetrate the endoplasmic reticulum (ER) membrane to reach the cytosol en route to the nucleus for infection. To penetrate the ER membrane, the prototype PyV simian virus 40 (SV40) induces formation of ER-escape sites, called foci, composed of repeating units of multi-tubular ER junctions where the virus is thought to exit. How SV40 triggers formation of the ER-foci harboring these multi-tubular ER junctions is unclear. Here, we show that the ER morphogenic atlastin 2 (ATL2) and ATL3 membrane proteins play critical roles in SV40 infection. Mechanistically, ATL3 mobilizes to the ER-foci where it deploys its GTPase-dependent membrane fusion activity to promote formation of multi-tubular ER junctions within the ER-foci. ATL3 also engages an SV40-containing membrane penetration complex. By contrast, ATL2 does not reorganize to the ER-foci. Instead, it supports the reticular ER morphology critical for the integrity of the ATL3-dependent membrane complex. Our findings illuminate how two host factors play distinct roles in the formation of an essential membrane penetration site for a non-enveloped virus. IMPORTANCE Membrane penetration by non-enveloped viruses, a critical infection step, remains enigmatic. The non-enveloped PyV simian virus 40 (SV40) penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol en route for infection. During ER-to-cytosol membrane penetration, SV40 triggers formation of ER-associated structures (called ER-foci) that function as the membrane penetration sites. Here, we discover a role of the ATL ER membrane proteins-known to shape the ER morphology-during SV40-induced ER-foci formation. These findings illuminate how a non-enveloped virus hijacks host components to construct a membrane penetration structure.

Keywords:  endoplasmic reticulum; entry; non-enveloped virus; simian virus 40

DOI:  https://doi.org/10.1128/jvi.00756-23
Cell Mol Immunol. 2023 Aug 15.

MAVS-loaded unanchored Lys63-linked polyubiquitin chains activate the RIG-I-MAVS signaling cascade.

Feng Liu, Wanxin Zhuang, Bin Song, Yuan Yang, Junqi Liu, Yi Zheng, Bingyu Liu, Jie Zheng, Wei Zhao, Chengjiang Gao.

  The adaptor molecule MAVS forms prion-like aggregates to govern the RIG-I-like receptor (RLR) signaling cascade. Lys63 (K63)-linked polyubiquitination is critical for MAVS aggregation, yet the underlying mechanism and the corresponding E3 ligases and deubiquitinating enzymes (DUBs) remain elusive. Here, we found that the K63-linked polyubiquitin chains loaded on MAVS can be directly recognized by RIG-I to initiate RIG-I-mediated MAVS aggregation with the prerequisite of the CARDRIG-I-CARDMAVS interaction. Interestingly, many K63-linked polyubiquitin chains attach to MAVS via an unanchored linkage. We identified Ube2N as a major ubiquitin-conjugating enzyme for MAVS and revealed that Ube2N cooperates with the E3 ligase Riplet and TRIM31 to promote the unanchored K63-linked polyubiquitination of MAVS. In addition, we identified USP10 as a direct DUB that removes unanchored K63-linked polyubiquitin chains from MAVS. Consistently, USP10 attenuates RIG-I-mediated MAVS aggregation and the production of type I interferon. Mice with a deficiency in USP10 show more potent resistance to RNA virus infection. Our work proposes a previously unknown mechanism for the activation of the RLR signaling cascade triggered by MAVS-attached unanchored K63-linked polyubiquitin chains and establishes the DUB USP10 and the E2:E3 pair Ube2N-Riplet/TRIM31 as a specific regulatory system for the unanchored K63-linked ubiquitination and aggregation of MAVS upon viral infection.

Keywords:  Aggregation; MAVS; RIG-I; USP10; Unanchored K63-linked polyubiquitin chains

DOI:  https://doi.org/10.1038/s41423-023-01065-2
Annu Rev Pharmacol Toxicol. 2023 Aug 16.

From Thalidomide to Rational Molecular Glue Design for Targeted Protein Degradation.

Vladas Oleinikovas, Pablo Gainza, Thomas Ryckmans, Bernhard Fasching, Nicolas H Thomä.

Thalidomide and its derivatives are powerful cancer therapeutics that are among the best-understood molecular glue degraders (MGDs). These drugs selectively reprogram the E3 ubiquitin ligase cereblon (CRBN) to commit target proteins for degradation by the ubiquitin-proteasome system. MGDs create novel recognition interfaces on the surface of the E3 ligase that engage in induced protein-protein interactions with neosubstrates. Molecular insight into their mechanism of action opens exciting opportunities to engage a plethora of targets through a specific recognition motif, the G-loop. Our analysis shows that current CRBN-based MGDs can in principle recognize over 2,500 proteins in the human proteome that contain a G-loop. We review recent advances in tuning the specificity between CRBN and its MGD-induced neosubstrates and deduce a set of simple rules that govern these interactions. We conclude that rational MGD design efforts will enable selective degradation of many more proteins, expanding this therapeutic modality to more disease areas. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 64 is January 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-pharmtox-022123-104147
Mol Cell. 2023 Aug 17. pii: S1097-2765(23)00563-4. [Epub ahead of print]83(16): 2976-2990.e9

E4 ubiquitin ligase promotes mitofusin turnover and mitochondrial stress response.

Vincent Anton, Ira Buntenbroich, Tânia Simões, Mariana Joaquim, Leonie Müller, Reinhard Buettner, Margarete Odenthal, Thorsten Hoppe, Mafalda Escobar-Henriques.

  Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.

Keywords:  CMT2A; Cdc48/p97; E4; Fzo1; MFN2; UBE4B; Ufd2; fusion; mitochondria; mitofusin; stress; ubiquitin

DOI:  https://doi.org/10.1016/j.molcel.2023.07.021
Chem Sci. 2023 Aug 16. 14(32): 8635-8643

A chemical probe unravels the reactive proteome of health-associated catechols.

Angela Weigert Muñoz, Kevin M Meighen-Berger, Stephan M Hacker, Matthias J Feige, Stephan A Sieber.

Catechol-containing natural products are common constituents of foods, drinks, and drugs. Natural products carrying this motif are often associated with beneficial biological effects such as anticancer activity and neuroprotection. However, the molecular mode of action behind these properties is poorly understood. Here, we apply a mass spectrometry-based competitive chemical proteomics approach to elucidate the target scope of catechol-containing bioactive molecules from diverse foods and drugs. Inspired by the protein reactivity of catecholamine neurotransmitters, we designed and synthesised a broadly reactive minimalist catechol chemical probe based on dopamine. Initial labelling experiments in live human cells demonstrated broad protein binding by the probe, which was largely outcompeted by its parent compound dopamine. Next, we investigated the competition profile of a selection of biologically relevant catechol-containing substances. With this approach, we characterised the protein reactivity and the target scope of dopamine and ten biologically relevant catechols. Strikingly, proteins associated with the endoplasmic reticulum (ER) were among the main targets. ER stress assays in the presence of reactive catechols revealed an activation of the unfolded protein response (UPR). The UPR is highly relevant in oncology and cellular resilience, which may provide an explanation of the health-promoting effects attributed to many catechol-containing natural products.

DOI: https://doi.org/10.1039/d3sc00888f
Mol Cell. 2023 Aug 17. pii: S1097-2765(23)00566-X. [Epub ahead of print]83(16): 2959-2975.e7

Molecular mechanism for activation of the 26S proteasome by ZFAND5.

Donghoon Lee, Yanan Zhu, Louis Colson, Xiaorong Wang, Siyi Chen, Emre Tkacik, Lan Huang, Qi Ouyang, Alfred L Goldberg, Ying Lu.

  Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

Keywords:  ZFAND5; muscle atrophy; proteasome activation; proteasomes; protein degradation; ubiquitin

DOI:  https://doi.org/10.1016/j.molcel.2023.07.023
Mol Cell. 2023 Aug 17. pii: S1097-2765(23)00550-6. [Epub ahead of print]83(16): 2840-2855

Control of mRNA fate by its encoded nascent polypeptide.

Markus Höpfler, Ramanujan S Hegde.

  Cells tightly regulate mRNA processing, localization, and stability to ensure accurate gene expression in diverse cellular states and conditions. Most of these regulatory steps have traditionally been thought to occur before translation by the action of RNA-binding proteins. Several recent discoveries highlight multiple co-translational mechanisms that modulate mRNA translation, localization, processing, and stability. These mechanisms operate by recognition of the nascent protein, which is necessarily coupled to its encoding mRNA during translation. Hence, the distinctive sequence or structure of a particular nascent chain can recruit recognition factors with privileged access to the corresponding mRNA in an otherwise crowded cellular environment. Here, we draw on both well-established and recent examples to provide a conceptual framework for how cells exploit nascent protein recognition to direct mRNA fate. These mechanisms allow cells to dynamically and specifically regulate their transcriptomes in response to changes in cellular states to maintain protein homeostasis.

Keywords:  mRNA decay; mRNA localization; nascent chain; protein biogenesis; ribosome; translational regulation

DOI:  https://doi.org/10.1016/j.molcel.2023.07.014
Cell Rep. 2023 Aug 16. pii: S2211-1247(23)01009-4. [Epub ahead of print]42(8): 112998

Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal.

Katarzyna M Grochowska, Marit Sperveslage, Rajeev Raman, Antonio V Failla, Dawid Głów, Christian Schulze, Laura Laprell, Boris Fehse, Michael R Kreutz.

  The complex morphology of neurons poses a challenge for proteostasis because the majority of lysosomal degradation machinery is present in the cell soma. In recent years, however, mature lysosomes were identified in dendrites, and a fraction of those appear to fuse with the plasma membrane and release their content to the extracellular space. Here, we report that dendritic lysosomes are heterogeneous in their composition and that only those containing lysosome-associated membrane protein (LAMP) 2A and 2B fuse with the membrane and exhibit activity-dependent motility. Exocytotic lysosomes dock in close proximity to GluN2B-containing N-methyl-D-aspartate-receptors (NMDAR) via an association of LAMP2B to the membrane-associated guanylate kinase family member SAP102/Dlg3. NMDAR-activation decreases lysosome motility and promotes membrane fusion. We find that chaperone-mediated autophagy is a supplier of content that is released to the extracellular space via lysosome exocytosis. This mechanism enables local disposal of aggregation-prone proteins like TDP-43 and huntingtin.

Keywords:  CMA; CP: Cell biology; CP: Neuroscience; GluN2B NMDAR; LAMP2; SAP102; dendrites; exocytosis; lysosomes

DOI:  https://doi.org/10.1016/j.celrep.2023.112998
J Cell Sci. 2023 Aug 15. pii: jcs259725. [Epub ahead of print]136(16):

Post-translational modifications of ATG8 proteins - an emerging mechanism of autophagy control.

Jose L Nieto-Torres, Sviatlana Zaretski, Tianhui Liu, Peter D Adams, Malene Hansen.

  Autophagy is a recycling mechanism involved in cellular homeostasis with key implications for health and disease. The conjugation of the ATG8 family proteins, which includes LC3B (also known as MAP1LC3B), to autophagosome membranes, constitutes a hallmark of the canonical autophagy process. After ATG8 proteins are conjugated to the autophagosome membranes via lipidation, they orchestrate a plethora of protein-protein interactions that support key steps of the autophagy process. These include binding to cargo receptors to allow cargo recruitment, association with proteins implicated in autophagosome transport and autophagosome-lysosome fusion. How these diverse and critical protein-protein interactions are regulated is still not well understood. Recent reports have highlighted crucial roles for post-translational modifications of ATG8 proteins in the regulation of ATG8 functions and the autophagy process. This Review summarizes the main post-translational regulatory events discovered to date to influence the autophagy process, mostly described in mammalian cells, including ubiquitylation, acetylation, lipidation and phosphorylation, as well as their known contributions to the autophagy process, physiology and disease.

Keywords:  ATG8; Autophagy; GABARAP; LC3; Phosphorylation; Post-translational modifications

DOI:  https://doi.org/10.1242/jcs.259725
Nat Commun. 2023 Aug 18. 14(1): 5031

Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired.

Wenjing Liang, Shakti Sagar, Rishith Ravindran, Rita H Najor, Justin M Quiles, Liguo Chi, Rachel Y Diao, Benjamin P Woodall, Leonardo J Leon, Erika Zumaya, Jason Duran, David M Cauvi, Antonio De Maio, Eric D Adler, Åsa B Gustafsson.

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

DOI: https://doi.org/10.1038/s41467-023-40680-5
Nat Commun. 2023 Aug 18. 14(1): 4683

Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation.

Satoshi Yamanaka, Hirotake Furihata, Yuta Yanagihara, Akihito Taya, Takato Nagasaka, Mai Usui, Koya Nagaoka, Yuki Shoya, Kohei Nishino, Shuhei Yoshida, Hidetaka Kosako, Masaru Tanokura, Takuya Miyakawa, Yuuki Imai, Norio Shibata, Tatsuya Sawasaki.

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

DOI: https://doi.org/10.1038/s41467-023-40385-9
J Biol Chem. 2023 Aug 16. pii: S0021-9258(23)02193-2. [Epub ahead of print] 105165

Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity.

Erik Walinda, Kenji Sugase, Naoki Ishii, Masahiro Shirakawa, Kazuhiro Iwai, Daichi Morimoto.

Attachment of polyubiquitin chains to proteins is a major post-translational modification in eukaryotes. Linear ubiquitin chain assembly complex (LUBAC), consisting of HOIP, HOIL-1L, and SHARPIN, specifically synthesizes "head-to-tail" polyubiquitin chains, which are linked via the N-terminal methionine α-amino- and C-terminal carboxylate of adjacent ubiquitin units and are thus commonly called "linear" (poly)ubiquitin chains. LUBAC-assembled linear polyubiquitin chains play key roles in immune signaling and suppression of cell death via NF-κB-dependent and -independent pathways and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear polyubiquitin via its NZF domain. Despite the fact that the structure of the bound form of the HOIL-1L NZF domain with linear diubiquitin is known, several aspects of the recognition specificity remain unexplained. Here we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear diubiquitin-bound state, while rejecting other potential ubiquitin species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear ubiquitin binding, suggesting that NZF tail flexibility might play an important role in the association reaction. Interactions between the NZF core and tail revealed conserved electrostatic contacts, which were greatly affected by charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear ubiquitin affinity, and molecular dynamics simulations illustrated how NZF core-tail interactions were disrupted by charge repulsion, weakening the integrity of the linear diubiquitin-bound conformation. The described molecular determinants of linear diubiquitin binding provide insight into the dynamic aspects of the ubiquitin code and hint at an integral function of the NZF domain in full-length HOIL-1L.

DOI: https://doi.org/10.1016/j.jbc.2023.105165
Chem Sci. 2023 Aug 16. 14(32): 8433-8447

Opportunities and challenges of protein-based targeted protein degradation.

Fangfang Shen, Laura M K Dassama.

In the 20 years since the first report of a proteolysis targeting chimeric (PROTAC) molecule, targeted protein degradation (TPD) technologies have attempted to revolutionize the fields of chemical biology and biomedicine by providing exciting research opportunities and potential therapeutics. However, they primarily focus on the use of small molecules to recruit the ubiquitin proteasome system to mediate target protein degradation. This then limits protein targets to cytosolic domains with accessible and suitable small molecule binding pockets. In recent years, biologics such as proteins and nucleic acids have instead been used as binders for targeting proteins, thereby expanding the scope of TPD platforms to include secreted proteins, transmembrane proteins, and soluble but highly disordered intracellular proteins. This perspective summarizes the recent TPD platforms that utilize nanobodies, antibodies, and other proteins as binding moieties to deplete challenging targets, either through the ubiquitin proteasome system or the lysosomal degradation pathway. Importantly, the perspective also highlights opportunities and remaining challenges of current protein-based TPD technologies.

DOI: https://doi.org/10.1039/d3sc02361c
Dis Model Mech. 2023 Aug 01. pii: dmm049762. [Epub ahead of print]16(8):

An interaction between OTULIN and SCRIB uncovers roles for linear ubiquitination in planar cell polarity.

Stephanie M Almeida, Sofiia Ivantsiv, Rieko Niibori, Wade H Dunham, Brooke A Green, Liang Zhao, Anne-Claude Gingras, Sabine P Cordes.

  Planar cell polarity (PCP) plays critical roles in developmental and homeostatic processes. Membrane presentation of PCP complexes containing Van Gogh-like (VANGL) transmembrane proteins is central to PCP and can be directed by the scaffold protein scribble (SCRIB). The role atypical linear ubiquitin (Met1-Ub) chains might play in PCP is unknown. Here, HEK293 cell-based interactomic analyses of the Met1-Ub deubiquitinase OTULIN revealed that OTULIN can interact with SCRIB. Moreover, Met1-Ub chains associated with VANGL2 and PRICKLE1, but not SCRIB, can direct VANGL2 surface presentation. Mouse embryos lacking Otulin showed variable neural tube malformations, including rare open neural tubes, a deficit associated with PCP disruption in mice. In Madin-Darby canine kidney cells, in which the enrichment of VANGL2-GFP proteins at cell-cell contacts represents activated PCP complexes, endogenous OTULIN was recruited to these sites. In the human MDA-MB-231 breast cancer cell model, OTULIN loss caused deficits in Wnt5a-induced filopodia extension and trafficking of transfected HA-VANGL2. Taken together, these findings support a role for linear (de)ubiquitination in PCP signaling. The association of Met1-Ub chains with PCP complex components offers new opportunities for integrating PCP signaling with OTULIN-dependent immune and inflammatory pathways.

Keywords:  Linear ubiquitin; Linear ubiquitin assembly complex; Mouse genetics; OTULIN; Planar cell polarity; Scribble

DOI:  https://doi.org/10.1242/dmm.049762
Redox Biol. 2023 Aug 09. pii: S2213-2317(23)00240-9. [Epub ahead of print]65 102839

The NRF2-p97-NRF2 negative feedback loop.

Aryatara Shakya, Pengfei Liu, Jack Godek, Nicholas W McKee, Matthew Dodson, Annadurai Anandhan, Aikseng Ooi, Joe G N Garcia, Max Costa, Eli Chapman, Donna D Zhang.

  p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients.

Keywords:  Arsenic; Cancer; Nrf2; Oxidative stress; Proteostasis; p97

DOI:  https://doi.org/10.1016/j.redox.2023.102839
bioRxiv. 2023 Aug 01. pii: 2023.07.31.551342. [Epub ahead of print]

Selective inhibition of hsp90 paralogs: Structure and binding studies uncover the role of helix 1 in Grp94-selective ligand binding.

Nanette L S Que, Paul M Seidler, Wen J Aw, Gabriela Chiosis, Daniel T Gewirth.

Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the structural elements that mediate each paralog's response to inhibitor binding. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, the 8-aryl moiety of PU-H36 is inserted into Site 2, a conditionally available side pocket, but in Hsp90 it occupies Site 1, a non-selective side pocket that is accessible in all hsp90 paralogs. The structure of Grp94 in complex with the non-selective PU-H71 shows only Site 1 binding. Large conformational shifts involving helices 1, 4 and 5 of the N-terminal domain of Grp94 are associated with the engagement of the Site 2 pocket for ligand binding. To understand the origins of Site 2 pocket engagement, we tested the binding of Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 of the Grp94 N-terminal domain is the discriminating element that allows for remodeling of the ATP binding pocket and exposure of the Site 2 selective pocket.

DOI: https://doi.org/10.1101/2023.07.31.551342
bioRxiv. 2023 Aug 04. pii: 2023.08.03.551843. [Epub ahead of print]

Parallel proteomics and phosphoproteomics defines starvation signal specific processes in cell quiescence.

Siyu Sun, Daniel Tranchina, David Gresham.

Cells arrest growth and enter a quiescent state upon nutrient deprivation. However, the molecular processes by which cells respond to different starvation signals to regulate exit from the cell division cycle and initiation of quiescence remains poorly understood. To study the role of protein expression and signaling in quiescence we combined temporal profiling of the proteome and phosphoproteome using stable isotope labeling with amino acids in cell culture (SILAC) in Saccharomyces cerevisiae (budding yeast). We find that carbon and phosphorus starvation signals activate quiescence through largely distinct remodeling of the proteome and phosphoproteome. However, increased expression of mitochondrial proteins is associated with quiescence establishment in response to both starvation signals. Deletion of the putative quiescence regulator RIM15 , which encodes a serine-threonine kinase, results in reduced survival of cells starved for phosphorus and nitrogen, but not carbon. However, we identified common protein phosphorylation roles for RIM15 in quiescence that are enriched for RNA metabolism and translation. We also find evidence for RIM15-mediated phosphorylation of some targets, including IGO1, prior to starvation consistent with a functional role for RIM15 in proliferative cells. Finally, our results reveal widespread catabolism of amino acids in response to nitrogen starvation, indicating widespread amino acid recycling via salvage pathways in conditions lacking environmental nitrogen. Our study defines an expanded quiescent proteome and phosphoproteome in yeast, and highlights the multiple coordinated molecular processes at the level of protein expression and phosphorylation that are required for quiescence.

DOI: https://doi.org/10.1101/2023.08.03.551843
PLoS Genet. 2023 Aug 16. 19(8): e1010848

Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import.

Salomé Nashed, Houssam El Barbry, Médine Benchouaia, Angélie Dijoux-Maréchal, Thierry Delaveau, Nadia Ruiz-Gutierrez, Lucie Gaulier, Déborah Tribouillard-Tanvier, Guillaume Chevreux, Stéphane Le Crom, Benoit Palancade, Frédéric Devaux, Elodie Laine, Mathilde Garcia.

N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.

DOI: https://doi.org/10.1371/journal.pgen.1010848
Biochem J. 2023 Aug 17. pii: BCJ20230119. [Epub ahead of print]

Development of an OTUD1 ubiquitin variant inhibitor.

Qi Liu, Evan Mallette, Hui Zheng, Wei Zhang.

  OTUD1 (Ovarian tumor domain-containing deubiquitinase 1) is a member of the OTU domain-containing deubiquitinase family of enzymes involved in immunoregulation and tumorigenesis pathways. OTUD1 is comprised of three distinct regions: an unstructured N-terminal region, an OTU-fold catalytic domain, and a ubiquitin-interacting motif (UIM) containing region. Enhanced enzymatic activity and a strong preference for K63-linked substrates are imparted by the UIM containing region. We used phage display with a ubiquitin variant (UbV) library to identify binders for OTUD1 lacking the unstructured N-terminal region (OTUD1OTU+UIM) in an attempt to identify inhibitors bridging the catalytic domain and the UIM containing region. Two UbVs were identified (UbVOD.1 and UbVOD.2) with high affinity and specificity for OTUD1. Of the UbVs identified, UbVOD.1 inhibited OTUD1 activity towards mono-Ub and K63-linked di-Ub substrates in vitro with single-digit nanomolar IC50 and potently inhibited deubiquitinase activity with poly-Ub chains of other linkages. In vivo expression of UbVOD.1 alone was unstable, however as a di-UbV, global deubiquitination and deubiquitinase activity with the OTUD1 substrate RIPK1 were inhibited. Herein we describe the development of molecular tools for exploring the activity of OTUD1 in a cellular context, towards protein-based therapeutics.

Keywords:  Deubiquitinases; UIM; inhibitor; phage display; protein engineering; ubiquitin variants

DOI:  https://doi.org/10.1042/BCJ20230119
Sci Adv. 2023 Aug 18. 9(33): eadg8190

Regulation of translation by ribosomal RNA pseudouridylation.

Yu Zhao, Jay Rai, Hong Li.

Pseudouridine is enriched in ribosomal, spliceosomal, transfer, and messenger RNA and thus integral to the central dogma. The chemical basis for how pseudouridine affects the molecular apparatus such as ribosome, however, remains elusive owing to the lack of structures without this natural modification. Here, we studied the translation of a hypopseudouridylated ribosome initiated by the internal ribosome entry site (IRES) elements. We analyzed eight cryo-electron microscopy structures of the ribosome bound with the Taura syndrome virus IRES in multiple functional states. We found widespread loss of pseudouridine-mediated interactions through water and long-range base pairings. In the presence of the translocase, eukaryotic elongation factor 2, and guanosine 5'-triphosphate hydrolysis, the hypopseudouridylated ribosome favors a rare unconducive conformation for decoding that is partially recouped in the ribosome population that remains modified at the P-site uridine. The structural principles learned establish the link between functional defects and modification loss and are likely applicable to other pseudouridine-associated processes.

DOI: https://doi.org/10.1126/sciadv.adg8190
Nat Chem Biol. 2023 Aug 17.

Gelation of cytoplasmic expanded CAG RNA repeats suppresses global protein synthesis.

Yuyin Pan, Junmei Lu, Xinran Feng, Shengyi Lu, Yi Yang, Guang Yang, Shudan Tan, Liang Wang, Pilong Li, Shouqing Luo, Boxun Lu.

RNA molecules with the expanded CAG repeat (eCAGr) may undergo sol-gel phase transitions, but the functional impact of RNA gelation is completely unknown. Here, we demonstrate that the eCAGr RNA may form cytoplasmic gel-like foci that are rapidly degraded by lysosomes. These RNA foci may significantly reduce the global protein synthesis rate, possibly by sequestering the translation elongation factor eEF2. Disrupting the eCAGr RNA gelation restored the global protein synthesis rate, whereas enhanced gelation exacerbated this phenotype. eEF2 puncta were significantly enhanced in brain slices from a knock-in mouse model and from patients with Huntington's disease, which is a CAG expansion disorder expressing eCAGr RNA. Finally, neuronal expression of the eCAGr RNA by adeno-associated virus injection caused significant behavioral deficits in mice. Our study demonstrates the existence of RNA gelation inside the cells and reveals its functional impact, providing insights into repeat expansion diseases and functional impacts of RNA phase transition.

DOI: https://doi.org/10.1038/s41589-023-01384-5
bioRxiv. 2023 Aug 04. pii: 2023.08.04.552011. [Epub ahead of print]

Protein interaction network analysis of mTOR signaling reveals modular organization.

Devin T Wehle, Carter S Bass, Josef Sulc, Ghayda Mirzaa, Stephen E P Smith.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation, and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (PI3Ks), protein kinases (AKT) and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex co-immunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after one hour, despite phosphorylation changes observed after only five minutes. Using small molecule inhibitors of PI3K, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed 'modules', that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.

DOI: https://doi.org/10.1101/2023.08.04.552011
Trends Cell Biol. 2023 Aug 14. pii: S0962-8924(23)00154-X. [Epub ahead of print]

Autophagy in intestinal epithelial cells prevents gut inflammation.

Shahar Telpaz, Shai Bel.

  Intestinal epithelial cells form the largest barrier in the body, separating us from the outside world. Here, we review recent findings that highlight the role of autophagy in the cell-intrinsic response of the epithelial cells to the harsh intestinal environment and how they shape host physiology.

Keywords:  autophagy; epithelium; inflammation; inflammatory bowel diseases; intestinal barrier; intestine

DOI:  https://doi.org/10.1016/j.tcb.2023.07.010
Traffic. 2023 Aug 14.

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum.

Anja J Engel, Steffen Paech, Markus Langhans, James L van Etten, Anna Moroni, Gerhard Thiel, Oliver Rauh.

  When the K+ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.

Keywords:  codon usage; effect of synonymous codon exchange; membrane protein sorting; transmembrane domain hydrophobicity

DOI:  https://doi.org/10.1111/tra.12915