bims-proteo 2024-07-21 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–07–21
forty papers selected by
Eric Chevet, INSERM

Phosphorylation of GCN2 by mTOR confers adaptation to conditions of hyper-mTOR activation under stress.
Ccr4-Not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy.
Dysregulation of Ceramide Metabolism Causes Phytoceramide-Dependent Induction of the Unfolded Protein Response.
Proteostasis Landscapes of Selective versus Poorly Responsive CFTR Variants Reveals Structural Vulnerabilities to Correction.
INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement.
Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.
Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms.
ATAD1 prevents clogging of TOM and damage caused by un-imported mitochondrial proteins.
Biochemical and Structural Consequences of NEDD8 Acetylation.
An E3 ligase and autophagy adaptor regulates tau proteostasis through nested phase separation.
ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites.
The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during Drosophila melanogaster oogenesis.
Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly.
Dual protection by Bcp1 and Rkm1 ensures incorporation of uL14 into pre-60S ribosomal subunits.
A quantitative pipeline to assess secretion of human leptin coding variants reveals mechanisms underlying leptin deficiencies.
eIF1 and eIF5 dynamically control translation start site fidelity.
Repression of mRNA translation initiation by GIGYF1 via disrupting the eIF3-eIF4G1 interaction.
Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.
Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.
Rational correction of pathogenic conformational defects in HTRA1.
Pseudophosphorylation of single residues of the J-domain of DNAJA2 regulates the holding/folding balance of the Hsc70 system.
A eukaryotic-like ubiquitination system in bacterial antiviral defence.
Inhibition of PERK mediated UPR acts as a switch for reversal of residual senescence and as senolytic therapy in glioblastoma.
Stoichiometry and architecture of the human pyruvate dehydrogenase complex.
Multiscale photocatalytic proximity labeling reveals cell surface neighbors on and between cells.
Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling.
DAP12 interacts with RER1 and is retained in the secretory pathway before assembly with TREM2.
The autophagy adaptor TRIAD3A promotes tau fibrillation by nested phase separation.
Visualizing the translation landscape in human cells at high resolution.
Binding and sensing diverse small molecules using shape-complementary pseudocycles.
Multi-step regulation of microRNA expression and secretion into small extracellular vesicles by insulin.
GRAIL1 stabilizes misfolded mutant p53 through a ubiquitin ligase-independent, chaperone regulatory function.
E3 ubiquitin ligase IPI1 controls rice immunity and flowering via both E3 ligase-dependent and -independent pathways.
Small-molecule probe for IBD risk variant GPR65 I231L alters cytokine signaling networks through positive allosteric modulation.
Sculpting conducting nanopore size and shape through de novo protein design.
RqcH supports survival in the absence of non-stop ribosome rescue factors.
Cell-selective proteomics reveal novel effectors secreted by an obligate intracellular bacterial pathogen.
Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response.
Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.
The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis to maintain translational repression of maternal mRNA and oocyte quality.

J Biol Chem. 2024 Jul 14. pii: S0021-9258(24)02076-3. [Epub ahead of print] 107575

Phosphorylation of GCN2 by mTOR confers adaptation to conditions of hyper-mTOR activation under stress.

Odai Darawshi, Olaya Yassin, Miri Shmuel, Ronald C Wek, S Jalil Mahdizadeh, Leif A Eriksson, Maria Hatzoglou, Boaz Tirosh.

Adaptation to shortage in free amino acids (AA) is mediated by two pathways, the integrated stress response (ISR) and the mechanistic target of rapamycin (mTOR). In response to reduced levels, primarily of leucine or arginine, mTOR in its complex 1 configuration (mTORC1) is suppressed leading to a decrease in translation initiation and elongation. The eIF2α kinase general control nonderepressible 2 (GCN2) is activated by uncharged tRNAs, leading to induction of the ISR in response to a broader range of AA shortage. ISR confers a reduced translation initiation, while promoting the selective synthesis of stress proteins, such as ATF4. To efficiently adapt to AA starvation, the two pathways are cross-regulated at multiple levels. Here we identified a new mechanism of ISR/mTORC1 crosstalk that optimizes survival under AA starvation, when mTORC1 is forced to remain active. mTORC1 activation during acute AA shortage, augmented ATF4 expression in a GCN2-dependent manner. Under these conditions, enhanced GCN2 activity was not dependent on tRNA sensing, inferring a different activation mechanism. We identified a labile physical interaction between GCN2 and mTOR that results in a phosphorylation of GCN2 on serine 230 by mTOR, which promotes GCN2 activity. When examined under prolonged AA starvation, GCN2 phosphorylation by mTOR promoted survival. Our data unveils an adaptive mechanism to AA starvation, when mTORC1 evades inhibition.

DOI: https://doi.org/10.1016/j.jbc.2024.107575
J Biol Chem. 2024 Jul 16. pii: S0021-9258(24)02083-0. [Epub ahead of print] 107582

Ccr4-Not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy.

Daniel L Johnson, Ravinder Kumar, David Kakhniashvili, Lawrence M Pfeffer, R Nicholas Laribee.

  The Ccr4-Not complex contains the poorly understood Not4 ubiquitin ligase that functions in transcription, mRNA decay, translation, proteostasis, and endolysosomal nutrient signaling. To gain further insight into the in vivo functions of the ligase, we performed quantitative proteomics in Saccharomyces cerevisiae using yeast cells lacking Not4, or cells overexpressing wild-type Not4 or an inactive Not4 mutant. Herein, we provide evidence that balanced Not4 activity maintains ribosomal protein (RP) homeostasis independent of changes to RP mRNA or known Not4 ribosomal substrates. Intriguingly, we also find that Not4 loss activates 40S ribosomal autophagy independently of canonical Atg7-dependent macroautophagy, indicating that microautophagy is responsible. We previously demonstrated that Ccr4-Not stimulates target of rapamycin complex 1 (TORC1) signaling, which activates RP expression and inhibits autophagy, by maintaining vacuole V-ATPase H+ pump activity. Importantly, combining Not4 deficient cells with a mutant that blocks vacuole H+ export fully restores RP expression and increases 40S RP autophagy efficiency. In contrast, restoring TORC1 activity alone fails to rescue either process, indicating that Not4 loss disrupts additional endolysosomal functions that regulate RP expression and 40S autophagy. Analysis of the Not4 regulated proteome reveals increases in endolysosomal and autophagy-related factors that functionally interact with Not4 to control RP expression and affect 40S autophagy. Collectively, our data indicate that balanced Ccr4-Not ubiquitin ligase signaling maintains RP homeostasis and inhibits 40S autophagy via the ligase's emerging role as an endolysosomal regulator.

Keywords:  Ccr4-Not; autophagy; lysosome; microautophagy; ribosome; ubiquitin ligase

DOI:  https://doi.org/10.1016/j.jbc.2024.107582
Mol Biol Cell. 2024 Jul 18. mbcE24030121

Dysregulation of Ceramide Metabolism Causes Phytoceramide-Dependent Induction of the Unfolded Protein Response.

Tamayanthi Rajakumar, Md Amin Hossain, Sylwia A Stopka, Yagmur Micoogullari, Jessie Ang, Nathalie Y R Agar, John Hanna.

The unfolded protein response (UPR) detects and mitigates the harmful effects of dysregulated endoplasmic reticulum (ER) function. The UPR has been best characterized as a protein quality control response, and the sole UPR sensor in yeast, Ire1, is known to detect misfolded ER proteins. However, recent work suggests the UPR can also sense diverse defects within the ER membrane, including increased fatty acid saturation and altered phospholipid abundance. These and other lipid-related stimuli have been referred to as lipid bilayer stress, and may be sensed independently through Ire1's transmembrane domain. Here we show that loss of Isc1, a phospholipase that catabolizes complex ceramides, causes UPR induction, even in the absence of exogenous stress. A series of chemical and genetic approaches identified a requirement for very long chain fatty acid (VLCFA)-containing phytoceramides for UPR induction. In parallel, comprehensive lipidomics analyses identified large increases in abundance of specific VLCFA-containing phytoceramides in the isc1Δ mutant. We failed to identify evidence of an accompanying defect in protein quality control or ER-associated protein degradation. These results extend our understanding of lipid bilayer stress in the UPR, and provide a foundation for mechanistic investigation of this fascinating intersection between ceramide metabolism, membrane homeostasis, and the UPR.

DOI: https://doi.org/10.1091/mbc.E24-03-0121
bioRxiv. 2024 Jul 11. pii: 2024.07.10.602964. [Epub ahead of print]

Proteostasis Landscapes of Selective versus Poorly Responsive CFTR Variants Reveals Structural Vulnerabilities to Correction.

Eli Fritz McDonald, Minsoo Kim, John A Olson, Jens Meiler, Lars Plate.

Cystic Fibrosis (CF) is a lethal genetic disorder caused by variants in CF transmembrane conductance regulator (CFTR). Many disease variants are treatable with corrector compounds, which enhance the folding and trafficking of CFTR. However, correctors fail to elicit a response for every CFTR variant. Approximately 3% of persons with CF harbor poorly responsive CFTR variants. Here, we reveal that a group of poorly responsive variants overlap with selectively responsive variants in a critical domain interface (nucleotide-binding domain 1/intracellular loop 4 - NBD1/ICL4). Affinity purification mass spectrometry proteomics was used to profile the protein homeostasis (proteostasis) changes of CFTR variants during corrector treatment to assess modulated interactions with protein folding and maturation pathways. Responsive variant interactions converged on similar proteostasis pathways during correction. In contrast, poorly responsive variants subtly diverged, revealing a partial restoration of protein quality control surveillance and a capacity to correct some mutations. Computational structural modeling showed that corrector VX-445 failed to confer enough NBD1 stability to poorly responsive variants. NBD1 secondary stabilizing mutations rescued poorly responsive variants, revealing structural vulnerabilities in NBD1 required for treating poor responders. Our study provides a framework for discerning the underlying protein quality control and structural defects of CFTR variants not reached with existing drugs. These insights can help expand therapeutics to all susceptible CFTR variants to enhance personalized medicine efforts.

DOI: https://doi.org/10.1101/2024.07.10.602964
bioRxiv. 2024 Jul 07. pii: 2024.07.06.602365. [Epub ahead of print]

INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement.

Cara R Schiavon, Yuning Wang, Jasmine W Feng, Stephanie Garrett, Tsung-Chang Sung, Yelena Dayn, Chunxin Wang, Richard J Youle, Omar A Quintero-Carmona, Gerald S Shadel, Uri Manor.

Proper regulation of organelle dynamics and inter-organelle contacts is critical for cellular health and function. Both the endoplasmic reticulum (ER) and actin cytoskeleton are known to regulate organelle dynamics, but how, when, and where these two subcellular components are coordinated to control organelle dynamics remains unclear. Here, we show that ER-associated actin consistently marks mitochondrial, endosomal, and lysosomal fission sites. We also show that actin polymerization by the ER-anchored isoform of the formin protein INF2 is a key regulator of the morphology and mobility of these organelles. Together, our findings establish a mechanism by which INF2-mediated polymerization of ER-associated actin at ER-organelle contacts regulates organelle dynamics.

DOI: https://doi.org/10.1101/2024.07.06.602365
J Biol Chem. 2024 Jul 16. pii: S0021-9258(24)02085-4. [Epub ahead of print] 107584

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

Eri Hirata, Ken-Taro Sakata, Grace I Dearden, Faria Noor, Indu Menon, George N Chiduza, Anant K Menon.

  The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

Keywords:  CDG; Ist2; MurJ; N-glycosylation; Nvj1; dolichol-linked oligosaccharide; endoplasmic reticulum; lipid droplet; scramblase; yeast

DOI:  https://doi.org/10.1016/j.jbc.2024.107584
Nat Chem. 2024 Jul 19.

Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms.

Hanjie Jiang, Bryant D Miller, Thibault Viennet, Hyojeon Kim, Kwangwoon Lee, Haribabu Arthanari, Philip A Cole.

Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation.

DOI: https://doi.org/10.1038/s41557-024-01576-z
Cell Rep. 2024 Jul 17. pii: S2211-1247(24)00802-7. [Epub ahead of print]43(8): 114473

ATAD1 prevents clogging of TOM and damage caused by un-imported mitochondrial proteins.

John Kim, Madeleine Goldstein, Lauren Zecchel, Ryan Ghorayeb, Christopher A Maxwell, Hilla Weidberg.

  Mitochondria require the constant import of nuclear-encoded proteins for proper functioning. Impaired protein import not only depletes mitochondria of essential factors but also leads to toxic accumulation of un-imported proteins outside the organelle. Here, we investigate the consequences of impaired mitochondrial protein import in human cells. We demonstrate that un-imported proteins can clog the mitochondrial translocase of the outer membrane (TOM). ATAD1, a mitochondrial ATPase, removes clogged proteins from TOM to clear the entry gate into the mitochondria. ATAD1 interacts with both TOM and stalled proteins, and its knockout results in extensive accumulation of mitochondrial precursors as well as decreased protein import. Increased ATAD1 expression contributes to improved fitness of cells with inefficient mitochondrial protein import. Overall, we demonstrate the importance of the ATAD1 quality control pathway in surveilling protein import and its contribution to cellular health.

Keywords:  AAA ATPase; ATAD1; CP: Cell biology; CP: Metabolism; TOM clogging; mitochondrial protein import; mitochondrial stress; protein quality control; proteotoxicity

DOI:  https://doi.org/10.1016/j.celrep.2024.114473
Chembiochem. 2024 Jul 18. e202400478

Biochemical and Structural Consequences of NEDD8 Acetylation.

Simon Maria Kienle, Tobias Schneider, Christine Bernecker, Janina Bracker, Andreas Marx, Michael Kovermann, Martin Scheffner, Katrin Stuber.

  Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase.

Keywords:  NEDD8; NMR spectroscopy; acetylation; genetic code expansion; protein modifications

DOI:  https://doi.org/10.1002/cbic.202400478
Nat Cell Biol. 2024 Jul 19.

An E3 ligase and autophagy adaptor regulates tau proteostasis through nested phase separation.

DOI: https://doi.org/10.1038/s41556-024-01466-z
Nat Commun. 2024 Jul 17. 15(1): 6008

ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites.

Luciana Renna, Giovanni Stefano, Maria Paola Puggioni, Sang-Jin Kim, Anastasiya Lavell, John E Froehlich, Graham Burkart, Stefano Mancuso, Christoph Benning, Federica Brandizzi.

The plant endoplasmic reticulum (ER) contacts heterotypic membranes at membrane contact sites (MCSs) through largely undefined mechanisms. For instance, despite the well-established and essential role of the plant ER-chloroplast interactions for lipid biosynthesis, and the reported existence of physical contacts between these organelles, almost nothing is known about the ER-chloroplast MCS identity. Here we show that the Arabidopsis ER membrane-associated VAP27 proteins and the lipid-binding protein ORP2A define a functional complex at the ER-chloroplast MCSs. Specifically, through in vivo and in vitro association assays, we found that VAP27 proteins interact with the outer envelope membrane (OEM) of chloroplasts, where they bind to ORP2A. Through lipidomic analyses, we established that VAP27 proteins and ORP2A directly interact with the chloroplast OEM monogalactosyldiacylglycerol (MGDG), and we demonstrated that the loss of the VAP27-ORP2A complex is accompanied by subtle changes in the acyl composition of MGDG and PG. We also found that ORP2A interacts with phytosterols and established that the loss of the VAP27-ORP2A complex alters sterol levels in chloroplasts. We propose that, by interacting directly with OEM lipids, the VAP27-ORP2A complex defines plant-unique MCSs that bridge ER and chloroplasts and are involved in chloroplast lipid homeostasis.

DOI: https://doi.org/10.1038/s41467-024-50425-7
bioRxiv. 2024 Jul 05. pii: 2024.07.03.601952. [Epub ahead of print]

The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during Drosophila melanogaster oogenesis.

Samantha N Milano, Livia V Bayer, Julie J Ko, Caroline E Casella, Diana P Bratu.

Processing bodies (P-bodies) are cytoplasmic membrane-less organelles which host multiple mRNA processing events. While the fundamental principles of P-body organization are beginning to be elucidated in vitro , a nuanced understanding of how their assembly is regulated in vivo remains elusive. Here, we investigate the potential link between ER exit sites and P-bodies in Drosophila melanogaster egg chambers. Employing a combination of live and super-resolution imaging, we found that P-bodies associated with ER exit sites are larger and less mobile than cytoplasmic P-bodies, indicating that they constitute a distinct class of P-bodies which are more mature than their cytoplasmic counterparts. Moreover, we demonstrate that altering the composition of ER exit sites has differential effects on core P-body proteins (Me31B, Cup, and Trailer Hitch) suggesting a potential role for ER exit sites in P-body organization. We further show that in the absence of ER exit sites, P-body integrity is compromised and the stability and translational repression efficiency of the maternal mRNA, oskar , are reduced. Finally, we show that ER stress is communicated to P-bodies via ER exit sites, highlighting the pivotal role of ER exit sites as a bridge between membrane-bound and membrane-less organelles in ER stress response. Together, our data unveils the significance of ER exit sites not only in governing P-body organization, but also in facilitating inter-organellar communication during stress, potentially bearing implications for a variety of disease pathologies.

DOI: https://doi.org/10.1101/2024.07.03.601952
Nature. 2024 Jul 17.

Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly.

Jens Hör, Sharon G Wolf, Rotem Sorek.

Several immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defence1-5. Here we studied an antiphage defence system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fibre, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defence system release a mixture of partially assembled, tailless phage particles and fully assembled phages in which the central tail fibre is obstructed by the covalently attached ubiquitin-like protein. These phages show severely impaired infectivity, explaining how the defence system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.

DOI: https://doi.org/10.1038/s41586-024-07616-5
J Cell Biol. 2024 Aug 05. pii: e202306117. [Epub ahead of print]223(8):

Dual protection by Bcp1 and Rkm1 ensures incorporation of uL14 into pre-60S ribosomal subunits.

Min-Chi Yeh, Ning-Hsiang Hsu, Hao-Yu Chu, Cheng-Han Yang, Pang-Hung Hsu, Chi-Chi Chou, Jing-Ting Shie, Wei-Ming Lee, Meng-Chiao Ho, Kai-Yin Lo.

Eukaryotic ribosomal proteins contain extended regions essential for translation coordination. Dedicated chaperones stabilize the associated ribosomal proteins. We identified Bcp1 as the chaperone of uL14 in Saccharomyces cerevisiae. Rkm1, the lysine methyltransferase of uL14, forms a ternary complex with Bcp1 and uL14 to protect uL14. Rkm1 is transported with uL14 by importins to the nucleus, and Bcp1 disassembles Rkm1 and importin from uL14 simultaneously in a RanGTP-independent manner. Molecular docking, guided by crosslinking mass spectrometry and validated by a low-resolution cryo-EM map, reveals the correlation between Bcp1, Rkm1, and uL14, demonstrating the protection model. In addition, the ternary complex also serves as a surveillance point, whereas incorrect uL14 is retained on Rkm1 and prevented from loading to the pre-60S ribosomal subunits. This study reveals the molecular mechanism of how uL14 is protected and quality checked by serial steps to ensure its safe delivery from the cytoplasm until its incorporation into the 60S ribosomal subunit.

DOI: https://doi.org/10.1083/jcb.202306117
J Biol Chem. 2024 Jul 11. pii: S0021-9258(24)02063-5. [Epub ahead of print] 107562

A quantitative pipeline to assess secretion of human leptin coding variants reveals mechanisms underlying leptin deficiencies.

Harry J M Baird, Amber S Shun-Shion, Edson Mendes de Oliveira, Danièle Stalder, Lu Liang, Jessica Eden, Joseph E Chambers, I Sadaf Farooqi, David C Gershlick, Daniel J Fazakerley.

  The hormone leptin, primarily secreted by adipocytes, plays a crucial role in regulating whole-body energy homeostasis. Homozygous loss-of-function mutations in the leptin gene (LEP) cause hyperphagia and severe obesity, primarily through alterations in leptin's affinity for its receptor or changes in serum leptin concentrations. Although serum concentrations are influenced by various factors (e.g., gene expression, protein synthesis, stability in the serum), proper delivery of leptin from its site of synthesis in the endoplasmic reticulum via the secretory pathway to the extracellular serum is a critical step. However, the regulatory mechanisms and specific machinery involved in this trafficking route, particularly in the context of human LEP mutations, remain largely unexplored. We have employed the Retention Using Selective Hooks (RUSH) system to elucidate the secretory pathway of leptin. We have refined this system into a medium-throughput assay for examining the pathophysiology of a range of obesity-associated LEP variants. Our results reveal that leptin follows the default secretory pathway, with no additional regulatory steps identified prior to secretion. Through screening of leptin variants, we identified three mutations that lead to proteasomal degradation of leptin and one variant that significantly decreased leptin secretion, likely through aberrant disulfide bond formation. These observations have identified novel pathogenic effects of leptin variants, which can be informative for therapeutics and diagnostics. Finally, our novel quantitative screening platform can be adapted for other secreted proteins.

Keywords:  Leptin; adipokine; coding variants; secretion

DOI:  https://doi.org/10.1016/j.jbc.2024.107562
bioRxiv. 2024 Jul 13. pii: 2024.07.10.602410. [Epub ahead of print]

eIF1 and eIF5 dynamically control translation start site fidelity.

Rosslyn Grosely, Carlos Alvarado, Ivaylo P Ivanov, Oliver B Nicholson, Joseph D Puglisi, Thomas E Dever, Christopher P Lapointe.

Translation initiation defines the identity of a synthesized protein through selection of a translation start site on a messenger RNA. This process is essential to well-controlled protein synthesis, modulated by stress responses, and dysregulated in many human diseases. The eukaryotic initiation factors eIF1 and eIF5 interact with the initiator methionyl-tRNA i Met on the 40S ribosomal subunit to coordinate start site selection. Here, using single-molecule analysis of in vitro reconstituted human initiation combined with translation assays in cells, we examine eIF1 and eIF5 function. During translation initiation on a panel of RNAs, we monitored both proteins directly and in real time using single-molecule fluorescence. As expected, eIF1 loaded onto mRNAs as a component of the 43S initiation complex. Rapid (∼ 2 s) eIF1 departure required a translation start site and was delayed by alternative start sites and a longer 5' untranslated region (5'UTR). After its initial departure, eIF1 rapidly and transiently sampled initiation complexes, with more prolonged sampling events on alternative start sites. By contrast, eIF5 only transiently bound initiation complexes late in initiation immediately prior to association of eIF5B, which allowed joining of the 60S ribosomal subunit. eIF5 association required the presence of a translation start site and was inhibited and destabilized by alternative start sites. Using both knockdown and overexpression experiments in human cells, we validated that eIF1 and eIF5 have opposing roles during initiation. Collectively, our findings demonstrate how multiple eIF1 and eIF5 binding events control start-site selection fidelity throughout initiation, which is tuned in response to changes in the levels of both proteins.

DOI: https://doi.org/10.1101/2024.07.10.602410
Sci Adv. 2024 Jul 19. 10(29): eadl5638

Repression of mRNA translation initiation by GIGYF1 via disrupting the eIF3-eIF4G1 interaction.

Jung-Hyun Choi, Jun Luo, Geoffrey G Hesketh, Shuyue Guo, Angelos Pistofidis, Reese Jalal Ladak, Yuxin An, Parisa Naeli, Tommy Alain, T Martin Schmeing, Anne-Claude Gingras, Thomas Duchaine, Xu Zhang, Nahum Sonenberg, Seyed Mehdi Jafarnejad.

Viruses can selectively repress the translation of mRNAs involved in the antiviral response. RNA viruses exploit the Grb10-interacting GYF (glycine-tyrosine-phenylalanine) proteins 2 (GIGYF2) and eukaryotic translation initiation factor 4E (eIF4E) homologous protein 4EHP to selectively repress the translation of transcripts such as Ifnb1, which encodes the antiviral cytokine interferon-β (IFN-β). Herein, we reveal that GIGYF1, a paralog of GIGYF2, robustly represses cellular mRNA translation through a distinct 4EHP-independent mechanism. Upon recruitment to a target mRNA, GIGYF1 binds to subunits of eukaryotic translation initiation factor 3 (eIF3) at the eIF3-eIF4G1 interaction interface. This interaction disrupts the eIF3 binding to eIF4G1, resulting in transcript-specific translational repression. Depletion of GIGYF1 induces a robust immune response by derepressing IFN-β production. Our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism has profound implications for the host response to viral infections.

DOI: https://doi.org/10.1126/sciadv.adl5638
Nat Commun. 2024 Jul 13. 15(1): 5890

Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.

Meera Gupta, Alex N T Johnson, Edward R Cruz, Eli J Costa, Randi L Guest, Sophia Hsin-Jung Li, Elizabeth M Hart, Thao Nguyen, Michael Stadlmeier, Benjamin P Bratton, Thomas J Silhavy, Ned S Wingreen, Zemer Gitai, Martin Wühr.

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

DOI: https://doi.org/10.1038/s41467-024-49920-8
Cell Rep. 2024 Jul 16. pii: S2211-1247(24)00839-8. [Epub ahead of print]43(8): 114510

Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.

Ryan D Mouery, Kimberly Lukasik, Carolyn Hsu, Thomas Bonacci, Derek L Bolhuis, Xianxi Wang, C Allie Mills, E Drew Toomer, Owen G Canterbury, Kevin C Robertson, Timothy B Branigan, Nicholas G Brown, Laura E Herring, Stephanie L Gupton, Michael J Emanuele.

  Ubiquitination is an essential regulator of cell division. The kinase Polo-like kinase 1 (PLK1) promotes protein degradation at G2/M phase through the E3 ubiquitin ligase Skp1-Cul1-F box (SCF)βTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome is uncharacterized. Combining quantitative proteomics with pharmacologic PLK1 inhibition revealed a widespread, PLK1-dependent program of protein breakdown at G2/M. We validated many PLK1-regulated proteins, including substrates of the cell-cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct E3 ligases. We show that the protein-kinase-A-anchoring protein A-kinase anchor protein 2 (AKAP2) is cell-cycle regulated and that its mitotic degradation is dependent on the PLK1/βTrCP signaling axis. Expression of a non-degradable AKAP2 mutant resulted in actin defects and aberrant mitotic spindles, suggesting that AKAP2 degradation coordinates cytoskeletal organization during mitosis. These findings uncover PLK1's far-reaching role in shaping the mitotic proteome post-translationally and have potential implications in malignancies where PLK1 is upregulated.

Keywords:  AKAP2; CP: Cell biology; CP: Molecular biology; Cullin RING Ligase; Cyclin F; G2/M; Plk1; cell cycle; mitosis; protein degradation; ubiquitin; βTRCP

DOI:  https://doi.org/10.1016/j.celrep.2024.114510
Nat Commun. 2024 Jul 16. 15(1): 5944

Rational correction of pathogenic conformational defects in HTRA1.

Nathalie Beaufort, Linda Ingendahl, Melisa Merdanovic, Andree Schmidt, David Podlesainski, Tim Richter, Thorben Neumann, Michael Kuszner, Ingrid R Vetter, Patricia Stege, Steven G Burston, Anto Filipovic, Yasser B Ruiz-Blanco, Kenny Bravo-Rodriguez, Joel Mieres-Perez, Christine Beuck, Stephan Uebel, Monika Zobawa, Jasmin Schillinger, Rainer Malik, Katalin Todorov-Völgyi, Juliana Rey, Annabell Roberti, Birte Hagemeier, Benedikt Wefers, Stephan A Müller, Wolfgang Wurst, Elsa Sanchez-Garcia, Alexander Zimmermann, Xiao-Yu Hu, Tim Clausen, Robert Huber, Stefan F Lichtenthaler, Carsten Schmuck, Michael Giese, Markus Kaiser, Michael Ehrmann, Martin Dichgans.

Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.

DOI: https://doi.org/10.1038/s41467-024-49982-8
Protein Sci. 2024 Aug;33(8): e5105

Pseudophosphorylation of single residues of the J-domain of DNAJA2 regulates the holding/folding balance of the Hsc70 system.

Lorea Velasco-Carneros, Ganeko Bernardo-Seisdedos, Jean-Didier Maréchal, Oscar Millet, Fernando Moro, Arturo Muga.

  The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.

Keywords:  DNAJA2; Hsc70; J‐domain; chaperone; phosphorylation; protein folding

DOI:  https://doi.org/10.1002/pro.5105
Nature. 2024 Jul 17.

A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Lydia R Chambers, Qiaozhen Ye, Jiaxi Cai, Minheng Gong, Hannah E Ledvina, Huilin Zhou, Aaron T Whiteley, Raymond T Suhandynata, Kevin D Corbett.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

DOI: https://doi.org/10.1038/s41586-024-07730-4
Neuro Oncol. 2024 Jul 18. pii: noae134. [Epub ahead of print]

Inhibition of PERK mediated UPR acts as a switch for reversal of residual senescence and as senolytic therapy in glioblastoma.

Madhura Ketkar, Sanket Desai, Pranav Rana, Rahul Thorat, Sridhar Epari, Amit Dutt, Shilpee Dutt.

   BACKGROUND: Glioblastoma due to recurrence is clinically challenging with 10-15months overall survival. Previously we showed therapy induced senescence (TIS) in glioblastoma reverses causing recurrence. Here, we aim to delineate TIS reversal mechanism for potential therapeutic intervention to prevent GBM recurrence.
METHODS: Residual senescent (RS) and End of Residual Senescence (ERS) cells were captured from GBM patient-derived primary-cultures and cell lines mimicking clinical scenario. RNA-sequencing, transcript/protein validations, knock-down/inhibitor studies, ChIP RT-PCR, biochemical assays and IHCs were performed for mechanistics of TIS reversal. In vivo validations were conducted in GBM orthotopic mouse model.
RESULTS: Transcriptome analysis showed co-expression of ER stress-UPR and senescence associated secretory phenotype (SASP) with TIS induction and reversal. Robust SASP production and secretion by RS cells could induce senescence, ROS, DNA damage and ER stress in paracrine fashion independent of radiation. Neutralization of most significantly enriched cytokine from RS-secretome IL1β, suppressed SASP and delayed senescence reversal. Mechanistically, with SASP and massive protein accumulation in Endoplasmic reticulum, RS cells displayed stressed ER morphology, upregulated ER stress markers and PERK pathway activation via peIF2α-ATF4-CHOP which was spontaneously resolved in ERS. ChIP RT-PCR showed CHOP occupancy at CXCL8/IL8, CDKN1A/p21 and BCL2L1/BCLXL aiding survival. PERK knockdown/inhibition with GSK2606414 in combination with radiation led to sustained ER stress and senescence without SASP. PERKi in RS functioned as senolytic via apoptosis and prevented recurrence in vitro and in vivo ameliorating overall survival.
CONCLUSION: We demonstrate that PERK mediated UPR regulates senescence reversal and its inhibition can be exploited as potential seno-therapeutic option in glioblastoma.

Keywords:  ER stress; Glioblastoma; PERK; TIS reversal; Unfolded protein response

DOI:  https://doi.org/10.1093/neuonc/noae134
Sci Adv. 2024 Jul 19. 10(29): eadn4582

Stoichiometry and architecture of the human pyruvate dehydrogenase complex.

Rafal Zdanowicz, Pavel Afanasyev, Adam Pruška, Julian A Harrison, Christoph Giese, Daniel Boehringer, Alexander Leitner, Renato Zenobi, Rudi Glockshuber.

The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.

DOI: https://doi.org/10.1126/sciadv.adn4582
Science. 2024 Jul 19. 385(6706): eadl5763

Multiscale photocatalytic proximity labeling reveals cell surface neighbors on and between cells.

Zhi Lin, Kaitlin Schaefer, Irene Lui, Zi Yao, Andrea Fossati, Danielle L Swaney, Ajikarunia Palar, Andrej Sali, James A Wells.

Proximity labeling proteomics (PLP) strategies are powerful approaches to yield snapshots of protein neighborhoods. Here, we describe a multiscale PLP method with adjustable resolution that uses a commercially available photocatalyst, Eosin Y, which upon visible light illumination activates different photo-probes with a range of labeling radii. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor and orthogonally validated more than 20 neighbors using immunoassays and AlphaFold-Multimer prediction. We further profiled the protein neighborhoods of cell-cell synapses induced by bispecific T cell engagers and chimeric antigen receptor T cells. This integrated multiscale PLP platform maps local and distal protein networks on and between cell surfaces, which will aid in the systematic construction of the cell surface interactome, revealing horizontal signaling partners and reveal new immunotherapeutic opportunities.

DOI: https://doi.org/10.1126/science.adl5763
EMBO Rep. 2024 Jul 18.

Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling.

Misha Kalarikkal, Rimpi Saikia, Lizanne Oliveira, Yashashree Bhorkar, Akshay Lonare, Pallavi Varshney, Prathamesh Dhamale, Amitabha Majumdar, Jomon Joseph.

  ER-mitochondria contact sites (ERMCSs) regulate processes, including calcium homoeostasis, energy metabolism and autophagy. Previously, it was shown that during growth factor signalling, mTORC2/Akt gets recruited to and stabilizes ERMCSs. Independent studies showed that GSK3β, a well-known Akt substrate, reduces ER-mitochondria connectivity by disrupting the VAPB-PTPIP51 tethering complex. However, the mechanisms that regulate ERMCSs are incompletely understood. Here we find that annulate lamellae (AL), relatively unexplored subdomains of ER enriched with a subset of nucleoporins, are present at ERMCSs. Depletion of Nup358, an AL-resident nucleoporin, results in enhanced mTORC2/Akt activation, GSK3β inhibition and increased ERMCSs. Depletion of Rictor, a mTORC2-specific subunit, or exogenous expression of GSK3β, was sufficient to reverse the ERMCS-phenotype in Nup358-deficient cells. We show that growth factor-mediated activation of mTORC2 requires the VAPB-PTPIP51 complex, whereas, Nup358's association with this tether restricts mTORC2/Akt signalling and ER-mitochondria connectivity. Expression of a Nup358 fragment that is sufficient for interaction with the VAPB-PTPIP51 complex suppresses mTORC2/Akt activation and disrupts ERMCSs. Collectively, our study uncovers a novel role for Nup358 in controlling ERMCSs by modulating the mTORC2/Akt/GSK3β axis.

Keywords:  Annulate Lamellae; ER-mitochondria Contact Sites; GSK3β; Nucleoporins; mTORC2

DOI:  https://doi.org/10.1038/s44319-024-00204-8
Cell Mol Life Sci. 2024 Jul 15. 81(1): 302

DAP12 interacts with RER1 and is retained in the secretory pathway before assembly with TREM2.

Yanxia Liu, Sandra Theil, Melanie Ibach, Jochen Walter.

  DNAX-activating protein of 12 kDa (DAP12) is a transmembrane adapter protein expressed in lymphoid and myeloid lineage cells. It interacts with several immunoreceptors forming functional complexes that trigger intracellular signaling pathways. One of the DAP12 associated receptors is the triggering receptor expressed on myeloid cells 2 (TREM2). Mutations in both DAP12 and TREM2 have been linked to neurodegenerative diseases. However, mechanisms involved in the regulation of subcellular trafficking and turnover of these proteins are not well understood. Here, we demonstrate that proteasomal degradation of DAP12 is increased in the absence of TREM2. Interestingly, unassembled DAP12 is also retained in early secretory compartments, including the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC), thereby preventing its transport to the plasma membrane. We also show that unassembled DAP12 interacts with the retention in ER sorting receptor 1 (RER1). The deletion of endogenous RER1 decreases expression of functional TREM2-DAP12 complexes and membrane proximal signaling, and resulted in almost complete inhibition of phagocytic activity in THP-1 differentiated macrophage-like cells. These results indicate that RER1 acts as an important regulator of DAP12 containing immunoreceptor complexes and immune cell function.

Keywords:  Alzheimer disease; Microglia; Nasu-Hakola disease; Neurodegeneration; Neuroinflammation; Phosphorylation

DOI:  https://doi.org/10.1007/s00018-024-05298-w
Nat Cell Biol. 2024 Jul 15.

The autophagy adaptor TRIAD3A promotes tau fibrillation by nested phase separation.

Jiechao Zhou, Yang 'an Chuang, Javier Redding-Ochoa, Rongzhen Zhang, Alexander J Platero, Alexander H Barrett, Juan C Troncoso, Paul F Worley, Wenchi Zhang.

Multiple neurodegenerative diseases are characterized by aberrant proteinaceous accumulations of tau. Here, we report a RING-in-between-RING-type E3 ligase, TRIAD3A, that functions as an autophagy adaptor for tau. TRIAD3A(RNF216) is an essential gene with mutations causing age-progressive neurodegeneration. Our studies reveal that TRIAD3A E3 ligase catalyses mixed K11/K63 polyubiquitin chains and self-assembles into liquid-liquid phase separated (LLPS) droplets. Tau is ubiquitinated and accumulates within TRIAD3A LLPS droplets and, via LC3 interacting regions, targets tau for autophagic degradation. Unexpectedly, tau sequestered within TRIAD3A droplets rapidly converts to fibrillar aggregates without the transitional liquid phase of tau. In vivo studies show that TRIAD3A decreases the accumulation of phosphorylated tau in a tauopathy mouse model, and a disease-associated mutation of TRIAD3A increases accumulation of phosphorylated tau, exacerbates gliosis and increases pathological tau spreading. In human Alzheimer disease brain, TRIAD3A co-localizes with tau amyloid in multiple histological forms, suggesting a role in tau proteostasis. TRIAD3A is an autophagic adaptor that utilizes E3 ligase and LLPS as a mechanism to capture cargo and appears especially relevant to neurodegenerative diseases.

DOI: https://doi.org/10.1038/s41556-024-01461-4
bioRxiv. 2024 Jul 02. pii: 2024.07.02.601723. [Epub ahead of print]

Visualizing the translation landscape in human cells at high resolution.

Wei Zheng, Yuekang Zhang, Jimin Wang, Shuhui Wang, Pengxin Chai, Elizabeth J Bailey, Wangbiao Guo, Swapnil C Devarkar, Shenping Wu, Jianfeng Lin, Kai Zhang, Jun Liu, Ivan B Lomakin, Yong Xiong.

Obtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo-electron microscopy (cryo-EM). With this in situ cryo-EM approach we resolved a 2.19 Å consensus structure of the human 80S ribosome and unveiled its 21 distinct functional states, nearly all higher than 3 Å resolution. In contrast to in vitro studies, we identified protein factors, including SERBP1, EDF1 and NAC/3, not enriched on purified ribosomes. Most strikingly, we observed that SERBP1 binds to the ribosome in almost all translating and non-translating states to bridge the 60S and 40S ribosomal subunits. These newly observed binding sites suggest that SERBP1 may serve an important regulatory role in translation. We also uncovered a detailed interface between adjacent translating ribosomes which can form the helical polysome structure. Finally, we resolved high-resolution structures from cells treated with homoharringtonine and cycloheximide, and identified numerous polyamines bound to the ribosome, including a spermidine that interacts with cycloheximide bound at the E site of the ribosome, underscoring the importance of high-resolution in situ studies in the complex native environment. Collectively, our work represents a significant advancement in detailed structural studies within cellular contexts.

DOI: https://doi.org/10.1101/2024.07.02.601723
Science. 2024 Jul 19. 385(6706): 276-282

Binding and sensing diverse small molecules using shape-complementary pseudocycles.

Linna An, Meerit Said, Long Tran, Sagardip Majumder, Inna Goreshnik, Gyu Rie Lee, David Juergens, Justas Dauparas, Ivan Anishchenko, Brian Coventry, Asim K Bera, Alex Kang, Paul M Levine, Valentina Alvarez, Arvind Pillai, Christoffer Norn, David Feldman, Dmitri Zorine, Derrick R Hicks, Xinting Li, Mariana Garcia Sanchez, Dionne K Vafeados, Patrick J Salveson, Anastassia A Vorobieva, David Baker.

We describe an approach for designing high-affinity small molecule-binding proteins poised for downstream sensing. We use deep learning-generated pseudocycles with repeating structural units surrounding central binding pockets with widely varying shapes that depend on the geometry and number of the repeat units. We dock small molecules of interest into the most shape complementary of these pseudocycles, design the interaction surfaces for high binding affinity, and experimentally screen to identify designs with the highest affinity. We obtain binders to four diverse molecules, including the polar and flexible methotrexate and thyroxine. Taking advantage of the modular repeat structure and central binding pockets, we construct chemically induced dimerization systems and low-noise nanopore sensors by splitting designs into domains that reassemble upon ligand addition.

DOI: https://doi.org/10.1126/science.adn3780
Cell Rep. 2024 Jul 11. pii: S2211-1247(24)00820-9. [Epub ahead of print]43(7): 114491

Multi-step regulation of microRNA expression and secretion into small extracellular vesicles by insulin.

Marsel Lino, Ruben Garcia-Martin, Vitor Rosetto Muñoz, Gabriel Palermo Ruiz, Allah Nawaz, Bruna Brasil Brandão, Jonathan Dreyfus, Hui Pan, C Ronald Kahn.

  Tissues release microRNAs (miRNAs) in small extracellular vesicles (sEVs) including exosomes, which can regulate gene expression in distal cells, thus acting as modulators of local and systemic metabolism. Here, we show that insulin regulates miRNA secretion into sEVs from 3T3-L1 adipocytes and that this process is differentially regulated from cellular expression. Thus, of the 53 miRNAs upregulated and 66 miRNAs downregulated by insulin in 3T3-L1 sEVs, only 12 were regulated in parallel in cells. Insulin regulated this process in part by phosphorylating hnRNPA1, causing it to bind to AU-rich motifs in miRNAs, mediating their secretion into sEVs. Importantly, 43% of insulin-regulated sEV-miRNAs are implicated in obesity and insulin resistance. These include let-7 and miR-103, which we show regulate insulin signaling in AML12 hepatocytes. Together, these findings demonstrate an important layer to insulin's regulation of adipose biology and provide a mechanism of tissue crosstalk in obesity and other hyperinsulinemic states.

Keywords:  CP: Metabolism; CP: Molecular biology; RNA binding proteins; adipocyte; exosomes; extracellular miRNAs; insulin; insulin signaling; miRNA; small extracellular vesicles

DOI:  https://doi.org/10.1016/j.celrep.2024.114491
Mol Cancer Res. 2024 Jul 17.

GRAIL1 stabilizes misfolded mutant p53 through a ubiquitin ligase-independent, chaperone regulatory function.

Paramita Ray, Sangeeta Jaiswal, Daysha Ferrer-Torres, Zhuwen Wang, Derek Nancarrow, Meghan Curtin, May San Martinho, Shannon M Lacy, Srimathi Kasturirangan, Dafydd Thomas, Jason R Spence, Matthias C Truttmann, Kiran H Lagisetty, Theodore S Lawrence, Thomas D Wang, David G Beer, Dipankar Ray.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's (BE) progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL - gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and, stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for Hsp40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain, or a cell permeable peptide (Pep-J) encoding the above 10 amino acids, can bind and inhibit DNAJ-Hsp70 co-chaperone activity thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic BE and EAC cells and inhibit growth of patient-derived dysplastic BE organoids (PDOs) in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J is comparable to simvastatin, a cholesterol lowering drug, that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase independent, chaperone regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

DOI: https://doi.org/10.1158/1541-7786.MCR-24-0361
Dev Cell. 2024 Jul 12. pii: S1534-5807(24)00392-7. [Epub ahead of print]

E3 ubiquitin ligase IPI1 controls rice immunity and flowering via both E3 ligase-dependent and -independent pathways.

Hong Yi, Hui Shi, Wei Mao, Junjie Yin, Yanyan Ma, Li Xu, Linjie Jing, Min He, Xiaobo Zhu, Xiang Lu, Qing Xiong, Yongyan Tang, Qingqing Hou, Li Song, Long Wang, Weitao Li, Hong Yu, Xuewei Chen, Jiayang Li, Jing Wang.

  Immunity and flowering are energy-consuming processes. However, the mechanism underlying the balance between immunity and flowering remains to be elucidated. Here, we report that the E3 ligase ideal plant architecture 1 interactor 1 (IPI1) controls rice immunity and flowering via two different pathways, one dependent on and another independent of its E3 ligase activity. We found that IPI1, a RING-finger E3 ligase, interacts with another E3 ligase, AvrPiz-t-interacting protein 6 (APIP6), and protects APIP6 from degradation by preventing APIP6's self-ubiquitination. Stabilization of APIP6 by IPI1 requires no IPI1 E3 ligase activity and leads to degradation of APIP6 substrates via the ubiquitin-proteasome system (UPS). Meanwhile, IPI1 directly ubiquitinates OsELF3-1 and OsELF3-2, two homologs of EARLY FLOWERING3 (ELF3), targeting them for degradation via the 26S proteasome. IPI1 knockout plants display early flowering but compromised resistance to rice blast. Thus, IPI1 balances rice immunity and flowering via both E3 ligase-dependent and -independent pathways.

Keywords:  E3 ligase; IPI1; flowering time; plant immunity; rice; self-ubiquitination; ubiquitin-proteasome system

DOI:  https://doi.org/10.1016/j.devcel.2024.06.014
Sci Adv. 2024 Jul 19. 10(29): eadn2339

Small-molecule probe for IBD risk variant GPR65 I231L alters cytokine signaling networks through positive allosteric modulation.

Ilona Neale, Clark Reddy, Zher Yin Tan, Bihua Li, Partha P Nag, Joshua Park, Jihye Park, Kimberly L Carey, Daniel B Graham, Ramnik J Xavier.

The proton-sensing heterotrimeric guanine nucleotide-binding protein-coupled receptor GPR65 is expressed in immune cells and regulates tissue homeostasis in response to decreased extracellular pH, which occurs in the context of inflammation and tumorigenesis. Genome-wide association studies linked GPR65 to several autoimmune and inflammatory diseases such as multiple sclerosis and inflammatory bowel disease (IBD). The loss-of-function GPR65 I231L IBD risk variant alters cellular metabolism, impairs protective tissue functions, and increases proinflammatory cytokine production. Hypothesizing that a small molecule designed to potentiate GPR65 at subphysiological pH could decrease inflammatory responses, we found positive allosteric modulators of GPR65 that engage and activate both human and mouse orthologs of the receptor. We observed that the chemical probe BRD5075 alters cytokine and chemokine programs in dendritic cells, establishing that immune signaling can be modulated by targeting GPR65. Our investigation offers improved chemical probes to further interrogate the biology of human GPR65 and its clinically relevant genetic variants.

DOI: https://doi.org/10.1126/sciadv.adn2339
Science. 2024 Jul 19. 385(6706): 282-288

Sculpting conducting nanopore size and shape through de novo protein design.

Samuel Berhanu, Sagardip Majumder, Thomas Müntener, James Whitehouse, Carolin Berner, Asim K Bera, Alex Kang, Binyong Liang, Nasir Khan, Banumathi Sankaran, Lukas K Tamm, David J Brockwell, Sebastian Hiller, Sheena E Radford, David Baker, Anastassia A Vorobieva.

Transmembrane β-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane β-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.

DOI: https://doi.org/10.1126/science.adn3796
bioRxiv. 2024 Jul 12. pii: 2024.07.12.603306. [Epub ahead of print]

RqcH supports survival in the absence of non-stop ribosome rescue factors.

Katrina Callan, Cassidy R Prince, Heather A Feaga.

Ribosomes frequently translate truncated or damaged mRNAs due to the extremely short half-life of mRNAs in bacteria. When ribosomes translate mRNA that lacks a stop codon (non-stop mRNA), specialized pathways are required to rescue the ribosome from the 3' end of the mRNA. The most highly conserved non-stop rescue pathway is trans -translation, which is found in greater than 95% of bacterial genomes. In all Proteobacteria that have been studied, the alternative non-stop ribosome rescue factors, ArfA and ArfB, are essential in the absence of trans -translation. Here, we investigate the interaction between non-stop rescue pathways and RqcH, a ribosome quality control factor that is broadly conserved outside of Proteobacteria. RqcH does not act directly on non-stop ribosomes but adds a degron tag to stalled peptides that obstruct the large ribosomal subunit, which allows the stalled peptide to be cleared from the ribosome by peptidyl-tRNA hydrolase (PTH). We show that Bacillus subtilis can survive without trans -translation and BrfA (Bacillus ArfA homolog), due to the presence of RqcH. We also show that expression of RqcH and its helper protein RqcP rescues the synthetic lethality of Δ ssrA Δ arfA in Escherichia coli . These results suggest that non-stop ribosome complexes can be disassembled and then cleared because of the tagging activity of RqcH, and that this process is essential in the absence of non-stop ribosome rescue pathways. Moreover, we surveyed the conservation of ribosome rescue pathways in >14,000 bacterial genomes. Our analysis reveals a broad distribution of non-stop rescue pathways, especially trans -translation and RqcH, and a strong co-occurrence between the ribosome splitting factor MutS2 and RqcH. Altogether, our results support a role for RqcH in non-stop ribosome rescue and provide a broad survey of ribosome rescue pathways in diverse bacterial species.
Importance: Ribosome stalling on damaged mRNA is a major problem in bacteria. It is estimated that 2-4% of all translation reactions terminate with the ribosome stalled on a damaged mRNA lacking a stop codon. Mechanisms that rescue these ribosomes, such as trans -translation, are often essential for viability. We investigated the functional overlap between RqcH and the non-stop ribosome rescue systems (ArfA and trans -translation) that are present in both E. coli and B. subtilis . Since these two species are extremely distant relatives, our work is likely to have wider implications for understanding ribosome rescue in bacteria. Furthermore, we used a bioinformatics approach to examine the conservation and overlap of various ribosome rescue systems in >14,000 species throughout the bacterial domain. These results provide key insights into ribosome rescue in diverse phyla.

DOI: https://doi.org/10.1101/2024.07.12.603306
Nat Commun. 2024 Jul 18. 15(1): 6073

Cell-selective proteomics reveal novel effectors secreted by an obligate intracellular bacterial pathogen.

Allen G Sanderlin, Hannah Kurka Margolis, Abigail F Meyer, Rebecca L Lamason.

Pathogenic bacteria secrete protein effectors to hijack host machinery and remodel their infectious niche. Rickettsia spp. are obligate intracellular bacteria that can cause life-threatening disease, but their absolute dependence on the host cell has impeded discovery of rickettsial effectors and their host targets. We implemented bioorthogonal non-canonical amino acid tagging (BONCAT) during R. parkeri infection to selectively label, isolate, and identify effectors delivered into the host cell. As the first use of BONCAT in an obligate intracellular bacterium, our screen more than doubles the number of experimentally validated effectors for the genus. The seven novel secreted rickettsial factors (Srfs) we identified include Rickettsia-specific proteins of unknown function that localize to the host cytoplasm, mitochondria, and ER. We further show that one such effector, SrfD, interacts with the host Sec61 translocon. Altogether, our work uncovers a diverse set of previously uncharacterized rickettsial effectors and lays the foundation for a deeper exploration of the host-pathogen interface.

DOI: https://doi.org/10.1038/s41467-024-50493-9
EMBO J. 2024 Jul 15.

Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response.

Tekle Pauzaite, Niek Wit, Rachel V Seear, James A Nathan.

  The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

Keywords:  DUB; Deubiquitination; HIF; Hypoxia; USP43

DOI:  https://doi.org/10.1038/s44318-024-00166-6
Nat Commun. 2024 Jul 15. 15(1): 5939

Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.

Christian Hentrich, Mateusz Putyrski, Hanh Hanuschka, Waldemar Preis, Sarah-Jane Kellmann, Melissa Wich, Manuel Cavada, Sarah Hanselka, Victor S Lelyveld, Francisco Ylera.

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher "SpyLock". Using "BiLockCatcher", a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.

DOI: https://doi.org/10.1038/s41467-024-50296-y
bioRxiv. 2024 Jul 04. pii: 2024.07.01.601596. [Epub ahead of print]

The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis to maintain translational repression of maternal mRNA and oocyte quality.

Mohamed T Elaswad, Mingze Gao, Victoria E Tice, Cora G Bright, Grace M Thomas, Chloe Munderloh, Nicholas J Trombley, Christya N Haddad, Ulysses G Johnson, Ashley N Cichon, Jennifer A Schisa.

The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo C. elegans oogenesis model to determine the intrinsic properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and the Lsm protein, CAR-1. We demonstrate that MEX-3 undergoes liquid-liquid phase separation and appears to have intrinsic gel-like properties in vitro . We also identify novel roles for the CCT chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. CCT and actin also oppose the expansion of ER sheets that may promote ectopic condensation of RNA-binding proteins that are associated with de-repression of maternal mRNA. This regulatory network is essential to preserve oocyte quality, prevent infertility, and may have implications for understanding the role of hMex3 phase transitions in cancer.
Significance statement: The molecular mechanisms that regulate phase transitions of oogenic RNA-binding proteins are critical to elucidate but are not fully understood.We identify novel regulators of RNA-binding protein phase transitions in maturing oocytes that are required to maintain translational repression of maternal mRNAs and oocyte quality.This study is the first to elucidate a regulatory network involving the CCT chaperonin, actin, and the ER for phase transitions of RNA-binding proteins during oogenesis. Our findings for the conserved MEX-3 protein may also be applicable to better understanding the role of hMex3 phase transitions in cancer.

DOI: https://doi.org/10.1101/2024.07.01.601596