bims-proteo 2023-04-16 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒04‒16
24 papers selected by
Eric Chevet
INSERM

RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum.
The role of RNF149 in the pre-emptive quality control substrate ubiquitination.
Ubiquitinome profiling reveals in vivo UBE2D3 targets and implicates UBE2D3 in protein quality control.
STING recruits WIPI2 for autophagosome formation.
Circadian Oscillation Pattern of Endoplasmic Reticulum Quality Control (ERQC) Components in Human Embryonic Kidney HEK293 Cells.
Molecular basis of Climp63-mediated ER lumen spacing.
ATG5 provides host protection acting as a switch in the atg8ylation cascade between autophagy and secretion.
The active zone protein Clarinet regulates synaptic sorting of ATG-9 and presynaptic autophagy.
BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.
Structure of the human UBR5 E3 ubiquitin ligase.
Differential requirements for P stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress.
A regulatory circuit comprising the CBP and SIRT7 regulates FAM134B-mediated ER-phagy.
Lysosomal chloride transporter CLH-6 protects lysosome membrane integrity via cathepsin activation.
A real-time analysis of GFP unfolding by the AAA+ unfoldase PAN.
Deregulation of ER-mitochondria contact formation and mitochondrial calcium homeostasis mediated by VDAC in fragile X syndrome.
Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2.
Proteostasis and lysosomal quality control deficits in Alzheimer's disease neurons.
Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2.
Noncoding translation mitigation.
Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by Atf4 following optic nerve injury.
Asparagine starvation suppresses histone demethylation through iron depletion.
Translatome analysis of Tuberous Sclerosis Complex-1 patient-derived neural progenitor cells reveal rapamycin-dependent and independent alterations.
ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.
Targeted protein degrader development for cancer: advances, challenges, and opportunities.

Proc Natl Acad Sci U S A. 2023 Apr 18. 120(16): e2220340120

RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum.

Francesco Scavone, Samantha C Gumbin, Paul A Da Rosa, Ron R Kopito.

  Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs.

Keywords:  UFM1; UFMylation; endoplasmic reticulum; ribosome-associated quality control; ubiquitin

DOI:  https://doi.org/10.1073/pnas.2220340120
Commun Biol. 2023 04 08. 6(1): 385

The role of RNF149 in the pre-emptive quality control substrate ubiquitination.

Aster Legesse, Nathali Kaushansky, Ilana Braunstein, Haddas Saad, Gerardo Lederkremer, Ami Navon, Ariel Stanhill.

Protein quality control is a process in which a protein's folding status is constantly monitored. Mislocalized proteins (MLP), are processed by the various quality control pathways, as they are often misfolded due to inappropriate cellular surroundings. Polypeptides that fail to translocate into the ER due to an inefficient signal peptide, mutations or ER stress are recognized by the pre-emptive ER associated quality control (pEQC) pathway and degraded by the 26 S proteasome. In this report we reveal the role of RNF149, a membrane bound E3 ligase in the ubiquitination of known pEQC substrates. We demonstrate its selective binding only to non-translocated proteins and its association with known pEQC components. Impairment in RNF149 function increases translocation flux into the ER and manifests in a myeloproliferative neoplasm (MPN) phenotype, a pathological condition associated with pEQC impairment. Finally, the dynamic localization of RNF149 may provide a molecular switch to regulate pEQC during ER stress.

DOI: https://doi.org/10.1038/s42003-023-04763-9
Mol Cell Proteomics. 2023 Apr 12. pii: S1535-9476(23)00058-0. [Epub ahead of print] 100548

Ubiquitinome profiling reveals in vivo UBE2D3 targets and implicates UBE2D3 in protein quality control.

Zeliha Yalçin, Daniëlle Koot, Karel Bezstarosti, Daniel Salas-Lloret, Onno B Bleijerveld, Vera Boersma, Mattia Falcone, Román González-Prieto, Maarten Altelaar, Jeroen A A Demmers, Jacqueline J L Jacobs.

Ubiquitination has crucial roles in many cellular processes and dysregulation of ubiquitin machinery enzymes can result in various forms of pathogenesis. Cells only have a limited set of ubiquitin-conjugating (E2) enzymes to support the ubiquitination of many cellular targets. As individual E2 enzymes have many different substrates and interactions between E2 enzymes and their substrates can be transient, it is challenging to define all in vivo substrates of an individual E2 and the cellular processes it affects. Particularly challenging in this respect is UBE2D3, an E2 enzyme with promiscuous activity in vitro but less defined roles in vivo. Here, we set out to identify in vivo targets of UBE2D3 by using SILAC-based and label-free quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with the levels of proteins from metabolic pathways, in particular retinol metabolism, being the most affected. However, the impact of UBE2D3 depletion on the ubiquitinome was much more prominent. Interestingly, molecular pathways related to mRNA translation were the most affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control (RQC), is dependent on UBE2D3. We show by TULIP2 methodology that RPS10 and RPS20 are direct targets of UBE2D3 and demonstrate that UBE2D3's catalytic activity is required to ubiquitinate RPS10 in vivo. In addition, our data suggest that UBE2D3 acts at multiple levels in autophagic protein quality control (PQC). Collectively, our findings show that depletion of an E2 enzyme in combination with quantitative diGly-based ubiquitinome profiling is a powerful tool to identify new in vivo E2 substrates, as we have done here for UBE2D3. Our work provides an important resource for further studies on the in vivo functions of UBE2D3.

DOI: https://doi.org/10.1016/j.mcpro.2023.100548
Autophagy. 2023 Apr 13. 1-2

STING recruits WIPI2 for autophagosome formation.

Wei Wan, Wei Liu.

  Induction of autophagy is a primordial function of the cGAS-STING pathway. However, the molecular mechanisms regulating autophagosome formation during STING-induced autophagy remain largely unknown. Recently, we reported that STING directly interacts with WIPI2 to recruit WIPI2 onto STING-positive vesicles for LC3 lipidation and autophagosome formation. We found that STING and PtdIns3P competitively bind to the FRRG motif of WIPI2, resulting in a mutual inhibition between STING-induced and PtdIns3P-dependent autophagy. We also showed that STING-WIPI2 interaction is necessary for cells to clear cytoplasmic DNA and attenuate activated cGAS-STING signaling. In summary, by identifying the interaction between STING and WIPI2, our study revealed a mechanism that allows STING to bypass the canonical upstream machinery to induce autophagosome formation.Abbreviations: ATG: autophagy-related; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; cGAMP: cyclic GMP-AMP; cGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment; IRF3: interferon regulatory factor 3; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; STING: stimulator of interferon genes; TBK1: TANK-binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Keywords:  Autophagosome; STING; WIPI2; autophagy; cGAS

DOI:  https://doi.org/10.1080/15548627.2023.2202108
J Circadian Rhythms. 2023 ;21 1

Circadian Oscillation Pattern of Endoplasmic Reticulum Quality Control (ERQC) Components in Human Embryonic Kidney HEK293 Cells.

Yalcin Erzurumlu, Deniz Catakli, Hatice Kubra Dogan.

  The circadian clock regulates the "push-pull" of the molecular signaling mechanisms that arrange the rhythmic organization of the physiology to maintain cellular homeostasis. In mammals, molecular clock genes tightly arrange cellular rhythmicity. It has been shown that this circadian clock optimizes various biological processes, including the cell cycle and autophagy. Hence, we explored the dynamic crosstalks between the circadian rhythm and endoplasmic reticulum (ER)-quality control (ERQC) mechanisms. ER-associated degradation (ERAD) is one of the most important parts of the ERQC system and is an elaborate surveillance system that eliminates misfolded proteins. It regulates the steady-state levels of several physiologically crucial proteins, such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and the metastasis suppressor KAI1/CD82. However, the circadian oscillation of ERQC members and their roles in cellular rhythmicity requires further investigation. In the present study, we provided a thorough investigation of the circadian rhythmicity of the fifteen crucial ERQC members, including gp78, Hrd1, p97/VCP, SVIP, Derlin1, Ufd1, Npl4, EDEM1, OS9, XTP3B, Sel1L, Ufd2, YOD1, VCIP135 and FAM8A1 in HEK293 cells. We found that mRNA and protein accumulation of the ubiquitin conjugation, binding and processing factors, retrotranslocation-dislocation, substrate recognition and targeting components of ERQC exhibit oscillation under the control of the circadian clock. Moreover, we found that Hrd1 and gp78 have a possible regulatory function on Bmal1 turnover. The findings of the current study indicated that the expression level of ERQC components is fine-tuned by the circadian clock and major ERAD E3 ligases, Hrd1 and gp78, may influence the regulation of circadian oscillation by modulation of Bmal1 stability.

Keywords:  Circadian rhythm; Endoplasmic reticulum-associated degradation; Hrd1; Protein-Quality Control; gp78

DOI:  https://doi.org/10.5334/jcr.219
J Cell Sci. 2023 Apr 11. pii: jcs.260976. [Epub ahead of print]

Molecular basis of Climp63-mediated ER lumen spacing.

Lu Xu, Yun Xiang, Junjie Hu.

  The width of cisternal structures in the endoplasmic reticulum (ER) is maintained by ER-resident protein Climp63. Self-association of the Climp63 luminal domain (LD), even though moderate, plays a key role in shaping ER sheets. However, the molecular basis of luminal spacing remains elusive. Here, we analyze the homotypic interactions of the Climp63 LD using AI-predicted structures. The LD is highly alpha-helical, with a flexible leading helix followed by a five-helix bundle (5HB). Charge-based trans associations are formed between the tip of the 5HB and the C-terminus of the LD, consistent with generating a width of ∼50 nm for ER sheets. The leading helix of the LD is dispensable for homotypic interactions, but packing of the 5HB regulates self-association. The density of Climp63, likely reflecting the strength of cis interactions, influences the ER width, which is maintained by trans interactions. These results indicate that a general principle in maintaining membrane tethering is multi-modular self-association.

Keywords:  Endoplasmic reticulum; Membrane tethering; Structural prediction

DOI:  https://doi.org/10.1242/jcs.260976
Dev Cell. 2023 Apr 10. pii: S1534-5807(23)00130-2. [Epub ahead of print]

ATG5 provides host protection acting as a switch in the atg8ylation cascade between autophagy and secretion.

Fulong Wang, Ryan Peters, Jingyue Jia, Michal Mudd, Michelle Salemi, Lee Allers, Ruheena Javed, Thabata L A Duque, Masroor A Paddar, Einar S Trosdal, Brett Phinney, Vojo Deretic.

  ATG5 is a part of the E3 ligase directing lipidation of ATG8 proteins, a process central to membrane atg8ylation and canonical autophagy. Loss of Atg5 in myeloid cells causes early mortality in murine models of tuberculosis. This in vivo phenotype is specific to ATG5. Here, we show using human cell lines that absence of ATG5, but not of other ATGs directing canonical autophagy, promotes lysosomal exocytosis and secretion of extracellular vesicles and, in murine Atg5fl/fl LysM-Cre neutrophils, their excessive degranulation. This is due to lysosomal disrepair in ATG5 knockout cells and the sequestration by an alternative conjugation complex, ATG12-ATG3, of ESCRT protein ALIX, which acts in membrane repair and exosome secretion. These findings reveal a previously undescribed function of ATG5 in its host-protective role in murine experimental models of tuberculosis and emphasize the significance of the branching aspects of the atg8ylation conjugation cascade beyond the canonical autophagy.

Keywords:  ATG5; ESCRT; SARS-CoV-2; atg8ylation; autophagy; coronavirus; exosomes; lysosome; neutrophils; tuberculosis

DOI:  https://doi.org/10.1016/j.devcel.2023.03.014
PLoS Biol. 2023 Apr;21(4): e3002030

The active zone protein Clarinet regulates synaptic sorting of ATG-9 and presynaptic autophagy.

Zhao Xuan, Sisi Yang, Benjamin Clark, Sarah E Hill, Laura Manning, Daniel A Colón-Ramos.

Autophagy is essential for cellular homeostasis and function. In neurons, autophagosome biogenesis is temporally and spatially regulated to occur near presynaptic sites, in part via the trafficking of autophagy transmembrane protein ATG-9. The molecules that regulate autophagy by sorting ATG-9 at synapses remain largely unknown. Here, we conduct forward genetic screens at single synapses of C. elegans neurons and identify a role for the long isoform of the active zone protein Clarinet (CLA-1L) in regulating sorting of autophagy protein ATG-9 at synapses, and presynaptic autophagy. We determine that disrupting CLA-1L results in abnormal accumulation of ATG-9 containing vesicles enriched with clathrin. The ATG-9 phenotype in cla-1(L) mutants is not observed for other synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Through genetic analyses, we uncover the adaptor protein complexes that genetically interact with CLA-1 in ATG-9 sorting. We also determine that CLA-1L extends from the active zone to the periactive zone and genetically interacts with periactive zone proteins in ATG-9 sorting. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic autophagy.

DOI: https://doi.org/10.1371/journal.pbio.3002030
bioRxiv. 2023 Mar 28. pii: 2023.03.28.534572. [Epub ahead of print]

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Dongsheng Han, Samantha H Schaffner, Jonathan P Davies, Mary Lauren Benton, Lars Plate, Jared T Nordman.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3, a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased in H3K4me1 levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels, but also causes a decrease in H3K4me3 levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific demethylase 5 (KDM5/Lid), an enzyme that removes tri- and di- methyl marks from H3K4. Moreover, analysis of ChIP-seq data revealed that BRWD3 and KDM5 are significantly co- localized throughout the genome and that sites of H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

DOI: https://doi.org/10.1101/2023.03.28.534572
Structure. 2023 Mar 30. pii: S0969-2126(23)00093-X. [Epub ahead of print]

Structure of the human UBR5 E3 ubiquitin ligase.

Feng Wang, Qing He, Wenhu Zhan, Ziqi Yu, Efrat Finkin-Groner, Xiaojing Ma, Gang Lin, Huilin Li.

  The human UBR5 is a single polypeptide chain homology to E6AP C terminus (HECT)-type E3 ubiquitin ligase essential for embryonic development in mammals. Dysregulated UBR5 functions like an oncoprotein to promote cancer growth and metastasis. Here, we report that UBR5 assembles into a dimer and a tetramer. Our cryoelectron microscopy (cryo-EM) structures reveal that two crescent-shaped UBR5 monomers assemble head to tail to form the dimer, and two dimers bind face to face to form the cage-like tetramer with all four catalytic HECT domains facing the central cavity. Importantly, the N-terminal region of one subunit and the HECT of the other form an "intermolecular jaw" in the dimer. We show the jaw-lining residues are important for function, suggesting that the intermolecular jaw functions to recruit ubiquitin-loaded E2 to UBR5. Further work is needed to understand how oligomerization regulates UBR5 ligase activity. This work provides a framework for structure-based anticancer drug development and contributes to a growing appreciation of E3 ligase diversity.

Keywords:  E3 ligase; cryo-EM; structural biology; uniquitin-proteasome

DOI:  https://doi.org/10.1016/j.str.2023.03.010
Proc Natl Acad Sci U S A. 2023 Apr 18. 120(16): e2300521120

Differential requirements for P stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress.

Ritu Gupta, Alan G Hinnebusch.

  The General Amino Acid Control is a conserved response to amino acid starvation involving activation of protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2 (eIF2α) with attendant inhibition of global protein synthesis and increased translation of yeast transcriptional activator GCN4. Gcn2 can be activated by either amino acid starvation or conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it is unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast cells by starvation-independent (SI) ribosome-stalling: treatment with tigecycline, eliminating the sole gene encoding tRNAArgUCC, and depletion of translation termination factor eRF1. We further demonstrated requirements for the tRNA- and ribosome-binding domains of Gcn2, the positive effector proteins Gcn1/Gcn20, and the tethering of at least one of two distinct P1/P2 heterodimers to the uL10 subunit of the ribosomal P stalk, for detectable activation by SI-ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation elicited by starvation for histidine or branched-chain amino acids isoleucine/valine. These results indicate that Gcn2 activation has different requirements for the P stalk depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in amino acid-starved cells can functionally substitute for the P stalk in binding to the histidyl-tRNA synthetase-like domain of Gcn2 for eIF2α kinase activation by ribosomes stalled with A sites devoid of the eEF1A∙GTP∙aminoacyl-tRNA ternary complex.

Keywords:  P stalk; amino acid starvation; elongation; translation; yeast

DOI:  https://doi.org/10.1073/pnas.2300521120
J Cell Biol. 2023 May 01. pii: e202201068. [Epub ahead of print]222(5):

A regulatory circuit comprising the CBP and SIRT7 regulates FAM134B-mediated ER-phagy.

Xinyi Wang, Xiao Jiang, Boran Li, Jiahua Zheng, Jiansheng Guo, Lei Gao, Mengjie Du, Xialian Weng, Lin Li, She Chen, Jingzi Zhang, Lei Fang, Ting Liu, Liang Wang, Wei Liu, Dante Neculai, Qiming Sun.

Macroautophagy (autophagy) utilizes a serial of receptors to specifically recognize and degrade autophagy cargoes, including damaged organelles, to maintain cellular homeostasis. Upstream signals spatiotemporally regulate the biological functions of selective autophagy receptors through protein post-translational modifications (PTM) such as phosphorylation. However, it is unclear how acetylation directly controls autophagy receptors in selective autophagy. Here, we report that an ER-phagy receptor FAM134B is acetylated by CBP acetyltransferase, eliciting intense ER-phagy. Furthermore, FAM134B acetylation promoted CAMKII-mediated phosphorylation to sustain a mode of milder ER-phagy. Conversely, SIRT7 deacetylated FAM134B to temper its activities in ER-phagy to avoid excessive ER degradation. Together, this work provides further mechanistic insights into how ER-phagy receptor perceives environmental signals for fine-tuning of ER homeostasis and demonstrates how nucleus-derived factors are programmed to control ER stress by modulating ER-phagy.

DOI: https://doi.org/10.1083/jcb.202201068
J Cell Biol. 2023 Jun 05. pii: e202210063. [Epub ahead of print]222(6):

Lysosomal chloride transporter CLH-6 protects lysosome membrane integrity via cathepsin activation.

Qianqian Zhang, Yuan Li, Youli Jian, Meijiao Li, Xiaochen Wang.

Lysosomal integrity is vital for cell homeostasis, but the underlying mechanisms are poorly understood. Here, we identify CLH-6, the C. elegans ortholog of the lysosomal Cl-/H+ antiporter ClC-7, as an important factor for protecting lysosomal integrity. Loss of CLH-6 affects lysosomal degradation, causing cargo accumulation and membrane rupture. Reducing cargo delivery or increasing CPL-1/cathepsin L or CPR-2/cathepsin B expression suppresses these lysosomal defects. Inactivation of CPL-1 or CPR-2, like CLH-6 inactivation, affects cargo digestion and causes lysosomal membrane rupture. Thus, loss of CLH-6 impairs cargo degradation, leading to membrane damage of lysosomes. In clh-6(lf) mutants, lysosomes are acidified as in wild type but contain lower chloride levels, and cathepsin B and L activities are significantly reduced. Cl- binds to CPL-1 and CPR-2 in vitro, and Cl- supplementation increases lysosomal cathepsin B and L activities. Altogether, these findings suggest that CLH-6 maintains the luminal chloride levels required for cathepsin activity, thus facilitating substrate digestion to protect lysosomal membrane integrity.

DOI: https://doi.org/10.1083/jcb.202210063
J Magn Reson. 2023 Apr 05. pii: S1090-7807(23)00066-6. [Epub ahead of print]350 107431

A real-time analysis of GFP unfolding by the AAA+ unfoldase PAN.

Georg Krüger, John Kirkpatrick, Emilie Mahieu, Bruno Franzetti, Frank Gabel, Teresa Carlomagno.

  Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN-20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution.

Keywords:  AAA+ ATPase; GFP; PAN; Protein unfolding; Real-time NMR spectroscopy

DOI:  https://doi.org/10.1016/j.jmr.2023.107431
Dev Cell. 2023 Apr 10. pii: S1534-5807(23)00098-9. [Epub ahead of print]58(7): 597-615.e10

Deregulation of ER-mitochondria contact formation and mitochondrial calcium homeostasis mediated by VDAC in fragile X syndrome.

Ji Geng, Tejinder Pal Khaket, Jie Pan, Wen Li, Yan Zhang, Yong Ping, Maria Inmaculada Cobos Sillero, Bingwei Lu.

  Loss of fragile X messenger ribonucleoprotein (FMRP) causes fragile X syndrome (FXS), the most prevalent form of inherited intellectual disability. Here, we show that FMRP interacts with the voltage-dependent anion channel (VDAC) to regulate the formation and function of endoplasmic reticulum (ER)-mitochondria contact sites (ERMCSs), structures that are critical for mitochondrial calcium (mito-Ca2+) homeostasis. FMRP-deficient cells feature excessive ERMCS formation and ER-to-mitochondria Ca2+ transfer. Genetic and pharmacological inhibition of VDAC or other ERMCS components restored synaptic structure, function, and plasticity and rescued locomotion and cognitive deficits of the Drosophila dFmr1 mutant. Expressing FMRP C-terminal domain (FMRP-C), which confers FMRP-VDAC interaction, rescued the ERMCS formation and mito-Ca2+ homeostasis defects in FXS patient iPSC-derived neurons and locomotion and cognitive deficits in Fmr1 knockout mice. These results identify altered ERMCS formation and mito-Ca2+ homeostasis as contributors to FXS and offer potential therapeutic targets.

Keywords:  ER-mitochondria contact site; ERMCS; FMRP; FXS; VDAC; fragile X messenger ribonucleoprotein; fragile X syndrome; mito-Ca(2+) homeostasis; mitochondrial calcium homeostasis; voltage-dependent anion channel

DOI:  https://doi.org/10.1016/j.devcel.2023.03.002
Elife. 2023 Apr 11. pii: e84382. [Epub ahead of print]12

Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2.

Sang Hwa Kim, Kye D Nichols, Eric N Anderson, Yining Liu, Nandini Ramesh, Weiyan Jia, Connor J Kuerbis, Mark Scalf, Lloyd M Smith, Udai Bhan Pandey, Randal S Tibbetts.

  Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

Keywords:  D. melanogaster; cell biology; human; neuroscience

DOI:  https://doi.org/10.7554/eLife.84382
bioRxiv. 2023 Mar 27. pii: 2023.03.27.534444. [Epub ahead of print]

Proteostasis and lysosomal quality control deficits in Alzheimer's disease neurons.

Ching-Chieh Chou, Ryan Vest, Miguel A Prado, Joshua Wilson-Grady, Joao A Paulo, Yohei Shibuya, Patricia Moran-Losada, Ting-Ting Lee, Jian Luo, Steven P Gygi, Jeffery W Kelly, Daniel Finley, Marius Wernig, Tony Wyss-Coray, Judith Frydman.

The role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer's disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-β inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.

DOI: https://doi.org/10.1101/2023.03.27.534444
Nat Commun. 2023 Apr 08. 14(1): 1980

Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2.

Rui Li, Jingchen Shao, Young-June Jin, Haruya Kawase, Yu Ting Ong, Kerstin Troidl, Qi Quan, Lei Wang, Remy Bonnavion, Astrid Wietelmann, Francoise Helmbacher, Michael Potente, Johannes Graumann, Nina Wettschureck, Stefan Offermanns.

Activation of endothelial YAP/TAZ signaling is crucial for physiological and pathological angiogenesis. The mechanisms of endothelial YAP/TAZ regulation are, however, incompletely understood. Here we report that the protocadherin FAT1 acts as a critical upstream regulator of endothelial YAP/TAZ which limits the activity of these transcriptional cofactors during developmental and tumor angiogenesis by promoting their degradation. We show that loss of endothelial FAT1 results in increased endothelial cell proliferation in vitro and in various angiogenesis models in vivo. This effect is due to perturbed YAP/TAZ protein degradation, leading to increased YAP/TAZ protein levels and expression of canonical YAP/TAZ target genes. We identify the E3 ubiquitin ligase Mind Bomb-2 (MIB2) as a FAT1-interacting protein mediating FAT1-induced YAP/TAZ ubiquitination and degradation. Loss of MIB2 expression in endothelial cells in vitro and in vivo recapitulates the effects of FAT1 depletion and causes decreased YAP/TAZ degradation and increased YAP/TAZ signaling. Our data identify a pivotal mechanism of YAP/TAZ regulation involving FAT1 and its associated E3 ligase MIB2, which is essential for YAP/TAZ-dependent angiogenesis.

DOI: https://doi.org/10.1038/s41467-023-37671-x
Nature. 2023 Apr 12.

Noncoding translation mitigation.

Jordan S Kesner, Ziheng Chen, Peiguo Shi, Alexis O Aparicio, Michael R Murphy, Yang Guo, Aditi Trehan, Jessica E Lipponen, Yocelyn Recinos, Natura Myeku, Xuebing Wu.

Translation is pervasive outside of canonical coding regions, occurring in long noncoding RNAs, canonical untranslated regions and introns1-4, especially in ageing4-6, neurodegeneration5,7 and cancer8-10. Notably, the majority of tumour-specific antigens are results of noncoding translation11-13. Although the resulting polypeptides are often nonfunctional, translation of noncoding regions is nonetheless necessary for the birth of new coding sequences14,15. The mechanisms underlying the surveillance of translation in diverse noncoding regions and how escaped polypeptides evolve new functions remain unclear10,16-19. Functional polypeptides derived from annotated noncoding sequences often localize to membranes20,21. Here we integrate massively parallel analyses of more than 10,000 human genomic sequences and millions of random sequences with genome-wide CRISPR screens, accompanied by in-depth genetic and biochemical characterizations. Our results show that the intrinsic nucleotide bias in the noncoding genome and in the genetic code frequently results in polypeptides with a hydrophobic C-terminal tail, which is captured by the ribosome-associated BAG6 membrane protein triage complex for either proteasomal degradation or membrane targeting. By contrast, canonical proteins have evolved to deplete C-terminal hydrophobic residues. Our results reveal a fail-safe mechanism for the surveillance of unwanted translation from diverse noncoding regions and suggest a possible biochemical route for the preferential membrane localization of newly evolved proteins.

DOI: https://doi.org/10.1038/s41586-023-05946-4
bioRxiv. 2023 Mar 31. pii: 2023.03.29.534798. [Epub ahead of print]

Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by Atf4 following optic nerve injury.

Preethi Somasundaram, Madeline M Farley, Melissa A Rudy, David G Stefanoff, Malay Shah, Puneetha Goli, Jenny Heo, Shufang Wang, Nicholas M Tran, Trent A Watkins.

Previously we showed that neurodegeneration initiated by axonal insults depends in part on the stress-responsive kinase Perk (Larhammar et al., 2017). Here we show that Perk acts primarily through Activating Transcription Factor-4 (Atf4) to stimulate not only pro-apoptotic but also pro-regenerative responses following optic nerve injury. Using conditional knockout mice, we find an extensive Perk/Atf4-dependent transcriptional response that includes canonical Atf4 target genes and modest contributions by C/ebp homologous protein (Chop). Overlap with c-Jun-dependent transcription suggests interplay with a parallel stress pathway that couples regenerative and apoptotic responses. Accordingly, neuronal knockout of Atf4 recapitulates the neuroprotection afforded by Perk deficiency, and Perk or Atf4 knockout impairs optic axon regeneration enabled by disrupting the tumor suppressor Pten. These findings contrast with the transcriptional and functional consequences reported for CRISPR targeting of Atf4 or Chop and reveal an integral role for Perk/Atf4 in coordinating neurodegenerative and regenerative responses to CNS axon injury.

DOI: https://doi.org/10.1101/2023.03.29.534798
iScience. 2023 Apr 21. 26(4): 106425

Asparagine starvation suppresses histone demethylation through iron depletion.

Jie Jiang, Sankalp Srivastava, Sheng Liu, Gretchen Seim, Rodney Claude, Minghua Zhong, Sha Cao, Utpal Davé, Reuben Kapur, Amber L Mosley, Chi Zhang, Jun Wan, Jing Fan, Ji Zhang.

  Intracellular α-ketoglutarate is an indispensable substrate for the Jumonji family of histone demethylases (JHDMs) mediating most of the histone demethylation reactions. Since α-ketoglutarate is an intermediate of the tricarboxylic acid cycle and a product of transamination, its availability is governed by the metabolism of several amino acids. Here, we show that asparagine starvation suppresses global histone demethylation. This process is neither due to the change of expression of histone-modifying enzymes nor due to the change of intracellular levels of α-ketoglutarate. Rather, asparagine starvation reduces the intracellular pool of labile iron, a key co-factor for the JHDMs to function. Mechanistically, asparagine starvation suppresses the expression of the transferrin receptor to limit iron uptake. Furthermore, iron supplementation to the culture medium restores histone demethylation and alters gene expression to accelerate cell death upon asparagine depletion. These results suggest that suppressing iron-dependent histone demethylation is part of the cellular adaptive response to asparagine starvation.

Keywords:  Biological sciences; Epigenetics; Molecular biology; Molecular mechanism of gene regulation; Physiology

DOI:  https://doi.org/10.1016/j.isci.2023.106425
Res Sq. 2023 Mar 27. pii: rs.3.rs-2702044. [Epub ahead of print]

Translatome analysis of Tuberous Sclerosis Complex-1 patient-derived neural progenitor cells reveal rapamycin-dependent and independent alterations.

Inci Aksoylu, Pauline Martin, Francis Robert, Krzysztof Szkop, Nicholas Redmond, Shan Chen, Roberta Beauchamp, Irene Nobeli, Jerry Pelletier, Ola Larsson, Vijaya Ramesh.

Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD). The hamartin-tuberin (TSC1-TSC2) protein complex inactivates mechanistic target of rapamycin complex 1 (mTORC1) signaling, leading to increased protein synthesis via inactivation of translational repressor eIF4E-binding proteins (4E-BPs). In TSC1 -null neural progenitor cells (NPCs), we previously reported early ND phenotypic changes, including increased proliferation/altered neurite outgrowth, which were unaffected by mTORC1-inhibitor rapamycin. Here, using polysome-profiling to quantify translational efficiencies at a transcriptome-wide level, we observed numerous TSC1-dependent alterations in NPCs, largely recapitulated in post-mortem brains from ASD donors. Although rapamycin partially reversed TSC1-associated alterations, most neural activity/synaptic- or ASD-related genes remained insensitive but were inhibited by third-generation bi-steric, mTORC1-selective inhibitor RMC-6272, which also reversed altered ND phenotypes. Together these data reveal potential implications for treatment of TAND.

DOI: https://doi.org/10.21203/rs.3.rs-2702044/v1
Proc Natl Acad Sci U S A. 2023 Apr 18. 120(16): e2210418120

ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

Benjamin Mayro, Jacob P Hoj, Christian G Cerda-Smith, Haley M Hutchinson, Michael W Caminear, Hannah L Thrash, Peter S Winter, Suzanne E Wardell, Donald P McDonnell, Colleen Wu, Kris C Wood, Ann Marie Pendergast.

  The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Keywords:  ABL kinases; CPSF1; E3-ligase; HIF-1α; MYC

DOI:  https://doi.org/10.1073/pnas.2210418120
Trends Pharmacol Sci. 2023 May;pii: S0165-6147(23)00060-3. [Epub ahead of print]44(5): 303-317

Targeted protein degrader development for cancer: advances, challenges, and opportunities.

Yuan Fang, Shuhang Wang, Songzhe Han, Yizhou Zhao, Cunjing Yu, Huaqing Liu, Ning Li.

  Anticancer-targeted therapies inhibit various kinases implicated in cancer and have been used in clinical settings for decades. However, many cancer-related targets are proteins without catalytic activity and are difficult to target using traditional occupancy-driven inhibitors. Targeted protein degradation (TPD) is an emerging therapeutic modality that has expanded the druggable proteome for cancer treatment. With the entry of new-generation immunomodulatory drugs (IMiDs), selective estrogen receptor degraders (SERDs), and proteolysis-targeting chimera (PROTAC) drugs into clinical trials, the field of TPD has seen explosive growth in the past 10 years. Several challenges remain that need to be tackled to increase successful clinical translation of TPD drugs. We present an overview of the global landscape of clinical trials of TPD drugs over the past decade and summarize the clinical profiles of new-generation TPD drugs. In addition, we highlight the challenges and opportunities for the development of effective TPD drugs for future successful clinical translation.

Keywords:  IMiD; PROTAC; SERD; cancer; protein degradation

DOI:  https://doi.org/10.1016/j.tips.2023.03.003