bims-proteo 2024-04-14 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–04–14
thirty-six papers selected by
Eric Chevet, INSERM

Massive ER protein disposal by reticulophagy receptors and selective disposal by TOLLIP.
Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.
COPII with ALG2 and ESCRTs control lysosome-dependent microautophagy of ER exit sites.
Noncanonical assembly, neddylation and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex.
Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.
ARV1 deficiency induces lipid bilayer stress and enhances rDNA stability by activating the unfolded protein response in Saccharomyces cerevisiae.
Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.
The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase.
Systematic HOIP interactome profiling reveals critical roles of linear ubiquitination in tissue homeostasis.
Systematic investigation of the trafficking of glycoproteins on the cell surface.
Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons.
WHAMM functions in kidney reabsorption and polymerizes actin to promote autophagosomal membrane closure and cargo sequestration.
Mechanism of autocatalytic activation during proteasome assembly.
Applications of protein ubiquitylation and deubiquitylation in drug discovery.
A Hollow TFG Condensate Spatially Compartmentalizes the Early Secretory Pathway.
A cilia-bound unconventional secretory pathway for Drosophila odorant receptors.
Discovery and Validation of a Novel Inhibitor of HYPE-mediated AMPylation.
ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.
Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Airway Neutrophilia.
Structural basis of translation inhibition by a valine tRNA-derived fragment.
Deep learning to decode sites of RNA translation in normal and cancerous tissues.
Discovery and Characterization of a Novel Cereblon-Recruiting PRC1 Bridged PROTAC Degrader.
Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency.
NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation.
XBP1s activates METTL3/METTL14 for ER-phagy and paclitaxel sensitivity regulation in breast cancer.
The Endo-Lysosomal Damage Response.
Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding.
An RNA-dependent and phase-separated active subnuclear compartment safeguards repressive chromatin domains.
Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.
A chemical proteomics approach for global mapping of functional lysines on cell surface of living cell.
The DNA Damage Response (DDR) landscape of endometrial cancer defines discrete disease subtypes and reveals therapeutic opportunities.
Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.
Phosphorylation of ELYS promotes its interaction with VAPB at decondensing chromosomes during mitosis.
The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.
MILIP Binding to tRNAs Promotes Protein Synthesis to Drive Triple-Negative Breast Cancer.
Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration.

Autophagy. 2024 Apr 11. 1-2

Massive ER protein disposal by reticulophagy receptors and selective disposal by TOLLIP.

Yuki Hayashi, Hidenori Ichijo.

  Proteostasis of the endoplasmic reticulum (ER) is maintained by coordinated action of two major catabolic pathways: proteasome-dependent ER-associated degradation (ERAD) and less characterized lysosomal pathways. Recent studies on ER-specific autophagy (termed "reticulophagy") have highlighted the importance of lysosomes for ER proteostasis. Key to this process are proteins termed reticulophagy receptors that connect ER fragments and Atg8-family proteins, facilitating the lysosomal degradation of both native and aberrant ER proteins in a relatively nonselective manner. In contrast, our recent work identified TOLLIP as a novel type of cargo receptor specifically dedicated to the lysosomal degradation of aberrant ER membrane proteins. The clients of TOLLIP include an engineered model substrate, which mimics an ER-retained aberrant membrane protein, and motor neuron disease-linked misfolded mutants of VAPB and BSCL2/Seipin. TOLLIP acts as a receptor to connect these aberrant ER membrane proteins and phosphatidylinositol-3-phosphate (PtdIns3P) by recognizing the former through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain, and the latter through its C2 domain. These interactions enable PtdIns3P-dependent vesicular trafficking of aberrant membrane proteins to lysosomes without promoting reticulophagic turnover of bulk ER.

Keywords:  ER stress; ER-phagy; ERAD; TOLLIP; motor neuron disease; reticulophagy

DOI:  https://doi.org/10.1080/15548627.2024.2340417
Nat Struct Mol Biol. 2024 Apr 10.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.

Frank Adolf, Jiale Du, Ellen A Goodall, Richard M Walsh, Shaun Rawson, Susanne von Gronau, J Wade Harper, John Hanna, Brenda A Schulman.

Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.

DOI: https://doi.org/10.1038/s41594-024-01268-9
Dev Cell. 2024 Apr 06. pii: S1534-5807(24)00195-3. [Epub ahead of print]

COPII with ALG2 and ESCRTs control lysosome-dependent microautophagy of ER exit sites.

Ya-Cheng Liao, Song Pang, Wei-Ping Li, Gleb Shtengel, Heejun Choi, Kathy Schaefer, C Shan Xu, Jennifer Lippincott-Schwartz.

  Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca2+ is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress.

Keywords:  ALG2; COPII; ER exit sites; ESCRTs; FIB-SEM; autophagy; cellular stress; lysosome; mTOR

DOI:  https://doi.org/10.1016/j.devcel.2024.03.027
Nat Struct Mol Biol. 2024 Apr 11.

Noncanonical assembly, neddylation and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex.

Daniel Horn-Ghetko, Linus V M Hopf, Ishita Tripathi-Giesgen, Jiale Du, Sebastian Kostrhon, D Tung Vu, Viola Beier, Barbara Steigenberger, J Rajan Prabu, Luca Stier, Elias M Bruss, Matthias Mann, Yue Xiong, Brenda A Schulman.

Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, 'cullin-RING' and 'RBR', are individually found in several hundred human E3 ligases, and collaborate with E2 enzymes to catalyze ubiquitylation. However, the vertebrate-specific CUL9 complex with RBX1 (also called ROC1), of interest due to its tumor suppressive interaction with TP53, uniquely encompasses both cullin-RING and RBR domains. Here, cryo-EM, biochemistry and cellular assays elucidate a 1.8-MDa hexameric human CUL9-RBX1 assembly. Within one dimeric subcomplex, an E2-bound RBR domain is activated by neddylation of its own cullin domain and positioning from the adjacent CUL9-RBX1 in trans. Our data show CUL9 as unique among RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects it from deneddylation. Substrates are recruited to various upstream domains, while ubiquitylation relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.

DOI: https://doi.org/10.1038/s41594-024-01257-y
Res Sq. 2024 Mar 28. pii: rs.3.rs-4013396. [Epub ahead of print]

Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.

Analisa DiFeo, Rita A Avelar, Riya Gupta, Grace Carvette, Felipe da Veiga Leprevost, Jose Colina, Jessica Teitel, Alexey Nesvizhskii, Caitlin O'connor, Maria Hatzoglou, Shirish Shenolikar, Peter Arvan, Goutham Narla.

The integrated stress response (ISR) regulates cell fate during conditions of stress by leveraging the cell's capacity to endure sustainable and efficient adaptive stress responses. Protein phosphatase 2A (PP2A) activity modulation has been shown to be successful in achieving both therapeutic efficacy and safety across various cancer models; however, the molecular mechanisms driving its selective antitumor effects remain unclear. Here, we show for the first time that ISR plasticity relies on PP2A activation to regulate drug response and dictate cellular fate under conditions of chronic stress. We demonstrate that genetic and chemical modulation of the PP2A leads to chronic proteolytic stress and triggers an ISR to dictate cell fate. More specifically, we uncovered that the PP2A-TFE3-ATF4 pathway governs ISR cell plasticity during endoplasmic reticular and cellular stress independent of the unfolded protein response. We further show that normal cells reprogram their genetic signatures to undergo ISR-mediated adaptation and homeostatic recovery thereby successfully avoiding toxicity following PP2A-mediated stress. Conversely, oncogenic specific cytotoxicity induced by chemical modulation of PP2A is achieved by activating chronic and irreversible ISR in cancer cells. Our findings propose that a differential response to chemical modulation of PP2A is determined by intrinsic ISR plasticity, providing a novel biological vulnerability to selectively induce cancer cell death and improve targeted therapeutic efficacy.

DOI: https://doi.org/10.21203/rs.3.rs-4013396/v1
J Biol Chem. 2024 Apr 06. pii: S0021-9258(24)01774-5. [Epub ahead of print] 107273

ARV1 deficiency induces lipid bilayer stress and enhances rDNA stability by activating the unfolded protein response in Saccharomyces cerevisiae.

Sujin Hong, Hyeon-Geun Lee, Won-Ki Huh.

  The stability of ribosomal DNA (rDNA) is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2 in Saccharomyces cerevisiae. Alongside proteostasis, rDNA stability is a crucial factor regulating the replicative lifespan (RLS) of S. cerevisiae. The unfolded protein response (UPR) is induced by misfolding of proteins or an imbalance of membrane lipid composition and is responsible for degrading misfolded proteins and restoring endoplasmic reticulum (ER) membrane homeostasis. Recent investigations have suggested that the UPR can extend the RLS of yeast by enhancing protein quality control mechanisms, but the relationship between the UPR and rDNA stability remains unknown. In this study, we found that the deletion of ARV1, which encodes an ER protein of unknown molecular function, activates the UPR by inducing lipid bilayer stress. In arv1Δ cells, the UPR and the cell wall integrity pathway are activated independently of each other, and the high osmolarity glycerol (HOG) pathway is activated in a manner dependent on Ire1, which mediates the UPR. Activated Hog1 translocates the stress response transcription factor Msn2 to the nucleus, where it promotes the expression of nicotinamidase Pnc1, a well-known Sir2 activator. Following Sir2 activation, rDNA silencing and rDNA stability are promoted. Furthermore, the loss of other ER proteins, such as Pmt1 or Bst1, and ER stress induced by tunicamycin or inositol depletion also enhance rDNA stability in a Hog1-dependent manner. Collectively, these findings suggest that the induction of the UPR enhances rDNA stability in S. cerevisiae by promoting the Msn2-Pnc1-Sir2 pathway in a Hog1-dependent manner.

Keywords:  Arv1; Saccharomyces cerevisiae; Sir2; high osmolarity glycerol (HOG); rDNA silencing; unfolded protein response (UPR)

DOI:  https://doi.org/10.1016/j.jbc.2024.107273
Cell Rep. 2024 Apr 10. pii: S2211-1247(24)00397-8. [Epub ahead of print]43(4): 114069

Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.

Vera Magg, Alessandro Manetto, Katja Kopp, Chia Ching Wu, Mohsen Naghizadeh, Doris Lindner, Lucy Eke, Julia Welsch, Stefan M Kallenberger, Johanna Schott, Volker Haucke, Nicolas Locker, Georg Stoecklin, Alessia Ruggieri.

  The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of growth arrest and DNA damage-inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We establish that the 3' untranslated region of PPP1R15A mRNA contains an active AU-rich element (ARE) recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a component of the transient ISR memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.

Keywords:  ARE; Brf1; CP: Cell biology; CP: Molecular biology; GADD34; PPP1R15A; TTP; ZFP36; integrated stress response; molecular memory; stress adaptation

DOI:  https://doi.org/10.1016/j.celrep.2024.114069
Elife. 2024 Apr 10. pii: RP92746. [Epub ahead of print]12

The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase.

Valentin Bohl, Nele Merret Hollmann, Tobias Melzer, Panagiotis Katikaridis, Lena Meins, Bernd Simon, Dirk Flemming, Irmgard Sinning, Janosch Hennig, Axel Mogk.

  Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during, e.g., food processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.

Keywords:  AAA protein; E. coli; biochemistry; chaperone; chemical biology; disaggregation; heat resistance; heat shock protein; proteostasis

DOI:  https://doi.org/10.7554/eLife.92746
Nat Commun. 2024 Apr 06. 15(1): 2974

Systematic HOIP interactome profiling reveals critical roles of linear ubiquitination in tissue homeostasis.

Yesheng Fu, Lei Li, Xin Zhang, Zhikang Deng, Ying Wu, Wenzhe Chen, Yuchen Liu, Shan He, Jian Wang, Yuping Xie, Zhiwei Tu, Yadi Lyu, Yange Wei, Shujie Wang, Chun-Ping Cui, Cui Hua Liu, Lingqiang Zhang.

Linear ubiquitination catalyzed by HOIL-1-interacting protein (HOIP), the key component of the linear ubiquitination assembly complex, plays fundamental roles in tissue homeostasis by executing domain-specific regulatory functions. However, a proteome-wide analysis of the domain-specific interactome of HOIP across tissues is lacking. Here, we present a comprehensive mass spectrometry-based interactome profiling of four HOIP domains in nine mouse tissues. The interaction dataset provides a high-quality HOIP interactome resource with an average of approximately 90 interactors for each bait per tissue. HOIP tissue interactome presents a systematic understanding of linear ubiquitination functions in each tissue and also shows associations of tissue functions to genetic diseases. HOIP domain interactome characterizes a set of previously undefined linear ubiquitinated substrates and elucidates the cross-talk among HOIP domains in physiological and pathological processes. Moreover, we show that linear ubiquitination of Integrin-linked protein kinase (ILK) decreases focal adhesion formation and promotes the detachment of Shigella flexneri-infected cells. Meanwhile, Hoip deficiency decreases the linear ubiquitination of Smad ubiquitination regulatory factor 1 (SMURF1) and enhances its E3 activity, finally causing a reduced bone mass phenotype in mice. Overall, our work expands the knowledge of HOIP-interacting proteins and provides a platform for further discovery of linear ubiquitination functions in tissue homeostasis.

DOI: https://doi.org/10.1038/s41467-024-47289-2
Mol Cell Proteomics. 2024 Apr 07. pii: S1535-9476(24)00051-3. [Epub ahead of print] 100761

Systematic investigation of the trafficking of glycoproteins on the cell surface.

Xing Xu, Kejun Yin, Ronghu Wu.

  Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.

Keywords:  Cell-surface glycoprotein; MS-based proteomics; N-Glycan maturity; N-glycosylation; Protein folding; Protein trafficking

DOI:  https://doi.org/10.1016/j.mcpro.2024.100761
Nat Commun. 2024 Apr 10. 15(1): 3113

Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons.

Xiaoting Zhou, You-Kyung Lee, Xianting Li, Henry Kim, Carlos Sanchez-Priego, Xian Han, Haiyan Tan, Suiping Zhou, Yingxue Fu, Kerry Purtell, Qian Wang, Gay R Holstein, Beisha Tang, Junmin Peng, Nan Yang, Zhenyu Yue.

Autophagy is a conserved, catabolic process essential for maintaining cellular homeostasis. Malfunctional autophagy contributes to neurodevelopmental and neurodegenerative diseases. However, the exact role and targets of autophagy in human neurons remain elusive. Here we report a systematic investigation of neuronal autophagy targets through integrated proteomics. Deep proteomic profiling of multiple autophagy-deficient lines of human induced neurons, mouse brains, and brain LC3-interactome reveals roles of neuronal autophagy in targeting proteins of multiple cellular organelles/pathways, including endoplasmic reticulum (ER), mitochondria, endosome, Golgi apparatus, synaptic vesicle (SV) for degradation. By combining phosphoproteomics and functional analysis in human and mouse neurons, we uncovered a function of neuronal autophagy in controlling cAMP-PKA and c-FOS-mediated neuronal activity through selective degradation of the protein kinase A - cAMP-binding regulatory (R)-subunit I (PKA-RI) complex. Lack of AKAP11 causes accumulation of the PKA-RI complex in the soma and neurites, demonstrating a constant clearance of PKA-RI complex through AKAP11-mediated degradation in neurons. Our study thus reveals the landscape of autophagy degradation in human neurons and identifies a physiological function of autophagy in controlling homeostasis of PKA-RI complex and specific PKA activity in neurons.

DOI: https://doi.org/10.1038/s41467-024-47440-z
Mol Biol Cell. 2024 Apr 10. mbcE24010025

WHAMM functions in kidney reabsorption and polymerizes actin to promote autophagosomal membrane closure and cargo sequestration.

Alyssa M Coulter, Valerie Cortés, Corey J Theodore, Rachel E Cianciolo, Ron Korstanje, Kenneth G Campellone.

The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display structural abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.

DOI: https://doi.org/10.1091/mbc.E24-01-0025
Nat Struct Mol Biol. 2024 Apr 10.

Mechanism of autocatalytic activation during proteasome assembly.

Benjamin Velez, Richard M Walsh, Shaun Rawson, Aida Razi, Lea Adams, Erignacio Fermin Perez, Fenglong Jiao, Marie Blickling, Tamayanthi Rajakumar, Darlene Fung, Lan Huang, John Hanna.

Many large molecular machines are too elaborate to assemble spontaneously and are built through ordered pathways orchestrated by dedicated chaperones. During assembly of the core particle (CP) of the proteasome, where protein degradation occurs, its six active sites are simultaneously activated via cleavage of N-terminal propeptides. Such activation is autocatalytic and coupled to fusion of two half-CP intermediates, which protects cells by preventing activation until enclosure of the active sites within the CP interior. Here we uncover key mechanistic aspects of autocatalytic activation, which proceeds through alignment of the β5 and β2 catalytic triad residues, respectively, with these triads being misaligned before fusion. This mechanism contrasts with most other zymogens, in which catalytic centers are preformed. Our data also clarify the mechanism by which individual subunits can be added in a precise, temporally ordered manner. This work informs two decades-old mysteries in the proteasome field, with broader implications for protease biology and multisubunit complex assembly.

DOI: https://doi.org/10.1038/s41594-024-01262-1
J Biol Chem. 2024 Apr 04. pii: S0021-9258(24)01765-4. [Epub ahead of print] 107264

Applications of protein ubiquitylation and deubiquitylation in drug discovery.

Yilin Chen, Haoan Xue, Jianping Jin.

  The ubiquitin-proteasome system (UPS) is the major machinery mediating specific protein turnover in eukaryotic cells. By ubiquitylating unwanted, damaged, or harmful proteins and driving their degradation, UPS is involved in many important cellular processes. Several new UPS-based technologies, including molecular glue degraders and PROTACs (Proteolysis-targeting chimeras) to promote protein degradation, and DUBTACs (deubiquitinase-targeting chimeras) to increase protein stability, have been developed. By specifically inducing the interactions between different ubiquitin ligases and targeted proteins that are not otherwise related, molecular glue degraders and PROTACs degrade targeted proteins via the ubiquitin-proteasome system; in contrast, by inducing the proximity of targeted proteins to deubiquitinases, DUBTACs are created to clear degradable polyubiquitin chains to stabilize targeted proteins. In this review, we summarize the recent research progress in molecular glue degraders, PROTACs, and DUBTACs and their applications. We discuss immunomodulatory drugs (IMiDs), sulfonamides, CDK-targeting molecular glue degraders, and new development of PROTACs. We also introduce the principle of DUBTAC and its applications. Finally, we propose a few future directions of these three technologies related to targeted protein homeostasis.

Keywords:  DUBTAC; Molecular glue; PROTAC; deubiquitination; ubiquitin; ubiquitylation

DOI:  https://doi.org/10.1016/j.jbc.2024.107264
bioRxiv. 2024 Mar 27. pii: 2024.03.26.586876. [Epub ahead of print]

A Hollow TFG Condensate Spatially Compartmentalizes the Early Secretory Pathway.

William R Wegeng, Savannah M Bogus, Miguel Ruiz, Sindy R Chavez, Khalid S M Noori, Ingrid R Niesman, Andreas M Ernst.

In the early secretory pathway, endoplasmic reticulum (ER) and Golgi membranes form a nearly spherical interface. In this ribosome-excluding zone, bidirectional transport of cargo coincides with a spatial segregation of anterograde and retrograde carriers by an unknown mechanism. We show that at physiological conditions, Trk-fused gene (TFG) self-organizes to form a hollow, anisotropic condensate that matches the dimensions of the ER-Golgi interface. Regularly spaced hydrophobic residues in TFG control the condensation mechanism and result in a porous condensate surface. We find that TFG condensates act as a molecular sieve, enabling molecules corresponding to the size of anterograde coats (COPII) to access the condensate interior while restricting retrograde coats (COPI). We propose that a hollow TFG condensate structures the ER-Golgi interface to create a diffusion-limited space for bidirectional transport. We further propose that TFG condensates optimize membrane flux by insulating secretory carriers in their lumen from retrograde carriers outside TFG cages.

DOI: https://doi.org/10.1101/2024.03.26.586876
BMC Biol. 2024 Apr 12. 22(1): 84

A cilia-bound unconventional secretory pathway for Drosophila odorant receptors.

Najat Dzaki, Mattias Alenius.

   BACKGROUND: Post-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway.
RESULTS: In the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity.
CONCLUSIONS: Our results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons.

Keywords:   Drosophila ; Grasp65; Odorant receptor; Olfactory sensory neurons; Soma to cilia trafficking; Unconventional secretion

DOI:  https://doi.org/10.1186/s12915-024-01877-2
Cell Stress Chaperones. 2024 Apr 08. pii: S1355-8145(24)00063-4. [Epub ahead of print]

Discovery and Validation of a Novel Inhibitor of HYPE-mediated AMPylation.

Ali Camara, Heerak Chugh, Alyssa George, Lukas Dolidze, Kevin Ryu, Katrina J Holly, Daniel P Flaherty, Seema Mattoo.

  AMPylation-the covalent transfer of an AMP from ATP onto a target protein-is catalyzed by the human enzyme HYPE/FicD to regulate its substrate, the heat shock chaperone BiP. HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the ER (endoplasmic reticulum) and mounting an UPR (unfolded protein response) in times of proteostatic imbalance. Thus, manipulating HYPE's enzymatic activity is a key therapeutic strategy towards the treatment of various protein misfolding diseases, including neuropathy and early onset diabetes associated with two recently identified clinical mutations of HYPE. Herein, we present an optimized, fluorescence polarization-based, high-throughput screening (HTS) assay to discover activators and inhibitors of HYPE-mediated AMPylation. After challenging our HTS assay with over 30,000 compounds, we discovered a novel AMPylase inhibitor, I2.10. We also determined a low micromolar IC50 for I2.10 and employed biorthogonal counter-screens to validate its efficacy against HYPE's AMPylation of BiP. Further, we report low cytotoxicity of I2.10 on human cell lines. We thus established an optimized, high-quality HTS assay amenable to tracking HYPE's enzymatic activity at scale, and provide the first novel small-molecule inhibitor capable of perturbing HYPE-directed AMPylation of BiP in vitro. Our HTS assay and I2.10 compound serve as a platform for further development of HYPE-specific small-molecule therapeutics.

Keywords:  AMPylation/adenylylation; BiP/GRP78/HSPA5; ER stress; HYPE/FICD; UPR; diabetes; drug discovery; fluorescence polarization; high-throughput screen; neurodegeneration; posttranslational modification

DOI:  https://doi.org/10.1016/j.cstres.2024.04.001
Traffic. 2024 Apr;25(4): e12933

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.

Kevin Ostacolo, Adrián López García de Lomana, Clémence Larat, Valgerdur Hjaltalin, Kristrun Yr Holm, Sigríður S Hlynsdóttir, Margaret Soucheray, Linda Sooman, Ottar Rolfsson, Nevan J Krogan, Eirikur Steingrimsson, Danielle L Swaney, Margret H Ogmundsdottir.

  Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

Keywords:  ATG7; ATG7(2); GTEx; PPI; autophagy; glycolysis; isoforms; lipidation; mitochondrial activity

DOI:  https://doi.org/10.1111/tra.12933
Am J Respir Cell Mol Biol. 2024 Apr 09.

Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Airway Neutrophilia.

Dandan Wu, Xing Zhang, Kourtney M Zimmerly, Ruoning Wang, Amanda Livingston, Takao Iwawaki, Ashok Kumar, Xiang Wu, Matthew Campen, Michael A Mandell, Meilian Liu, Xuexian O Yang.

  Heightened unfolded protein responses (UPRs) are associated with the risk for asthma, including severe asthma. Treatment-refractory severe asthma manifests a neutrophilic phenotype with TH17 responses. However, how UPRs participate in the deregulation of TH17 cells leading to neutrophilic asthma remains elusive. This study found that the UPR sensor IRE1 is induced in the murine lung with fungal asthma and is highly expressed in TH17 cells relative to naïve CD4+ T cells. Cytokine (e.g. IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by both human and mouse TH17 cells. Ern1 (encoding IRE1)-deficiency decreases the expression of ER stress factors and impairs the differentiation and cytokine secretion of TH17 cells. Genetic ablation of Ern1 leads to alleviated TH17 responses and airway neutrophilia in a fungal airway inflammation model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway in vivo and enhances TH17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPR-mediated secretory function of TH17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in TH17-biased TH2-low asthma.

Keywords:  IRE1; JAK2; TH17 cell; UPR; neutrophilia

DOI:  https://doi.org/10.1165/rcmb.2023-0424OC
Life Sci Alliance. 2024 Jun;pii: e202302488. [Epub ahead of print]7(6):

Structural basis of translation inhibition by a valine tRNA-derived fragment.

Yun Wu, Meng-Ting Ni, Ying-Hui Wang, Chen Wang, Hai Hou, Xing Zhang, Jie Zhou.

Translational regulation by non-coding RNAs is a mechanism commonly used by cells to fine-tune gene expression. A fragment derived from an archaeal valine tRNA (Val-tRF) has been previously identified to bind the small subunit of the ribosome and inhibit translation in Haloferax volcanii Here, we present three cryo-electron microscopy structures of Val-tRF bound to the small subunit of Sulfolobus acidocaldarius ribosomes at resolutions between 4.02 and 4.53 Å. Within these complexes, Val-tRF was observed to bind to conserved RNA-interacting sites, including the ribosomal decoding center. The binding of Val-tRF destabilizes helices h24, h44, and h45 and the anti-Shine-Dalgarno sequence of 16S rRNA. The binding position of this molecule partially overlaps with the translation initiation factor aIF1A and occludes the mRNA P-site codon. Moreover, we found that the binding of Val-tRF is associated with steric hindrance of the H69 base of 23S rRNA in the large ribosome subunit, thereby preventing 70S assembly. Our data exemplify how tRNA-derived fragments bind to ribosomes and provide new insights into the mechanisms underlying translation inhibition by Val-tRFs.

DOI: https://doi.org/10.26508/lsa.202302488
bioRxiv. 2024 Mar 25. pii: 2024.03.21.586110. [Epub ahead of print]

Deep learning to decode sites of RNA translation in normal and cancerous tissues.

Jim Clauwaert, Zahra McVey, Ramneek Gupta, Ian Yannuzzi, Gerben Menschaert, John R Prensner.

The biological process of RNA translation is fundamental to cellular life and has wide-ranging implications for human disease. Yet, accurately delineating the variation in RNA translation represents a significant challenge. Here, we develop RiboTIE, a transformer model-based approach to map global RNA translation. We find that RiboTIE offers unparalleled precision and sensitivity for ribosome profiling data. Application of RiboTIE to normal brain and medulloblastoma cancer samples enables high-resolution insights into disease regulation of RNA translation.

DOI: https://doi.org/10.1101/2024.03.21.586110
J Med Chem. 2024 Apr 12.

Discovery and Characterization of a Novel Cereblon-Recruiting PRC1 Bridged PROTAC Degrader.

Md Kabir, Lihuai Qin, Kaixiu Luo, Yan Xiong, Rebecca A Sidi, Kwang-Su Park, Jian Jin.

Bridged PROTAC is a novel protein complex degrader strategy that exploits the target protein's binding partner to degrade undruggable proteins by inducing proximity to an E3 ubiquitin ligase. In this study, we discovered for the first time that cereblon (CRBN) can be employed for the bridged PROTAC approach and report the first-in-class CRBN-recruiting and EED-binding polycomb repressive complex 1 (PRC1) degrader, compound 1 (MS181). We show that 1 induces preferential degradation of PRC1 components, BMI1 and RING1B, in an EED-, CRBN-, and ubiquitin-proteosome system (UPS)-dependent manner. Compound 1 also has superior antiproliferative activity in multiple metastatic cancer cell lines over EED-binding PRC2 degraders and can be efficacious in VHL-defective cancer cells. Altogether, compound 1 is a valuable chemical biology tool to study the role of PRC1 in cancer. Importantly, we show that CRBN can be utilized to develop bridged PROTACs, expanding the bridged PROTAC technology for degrading undruggable proteins.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00538
Nat Immunol. 2024 Apr 12.

Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency.

Hirotsugu Oda, Kalpana Manthiram, Pallavi Pimpale Chavan, Eva Rieser, Önay Veli, Öykü Kaya, Charles Rauch, Shuichiro Nakabo, Hye Sun Kuehn, Mariël Swart, Yanli Wang, Nisa Ilgim Çelik, Anne Molitor, Vahid Ziaee, Nasim Movahedi, Mohammad Shahrooei, Nima Parvaneh, Nasrin Alipour-Olyei, Raphael Carapito, Qin Xu, Silvia Preite, David B Beck, Jae Jin Chae, Michele Nehrebecky, Amanda K Ombrello, Patrycja Hoffmann, Tina Romeo, Natalie T Deuitch, Brynja Matthíasardóttir, James Mullikin, Hirsh Komarow, Jennifer Stoddard, Julie Niemela, Kerry Dobbs, Colin L Sweeney, Holly Anderton, Kate E Lawlor, Hiroyuki Yoshitomi, Dan Yang, Manfred Boehm, Jeremy Davis, Pamela Mudd, Davide Randazzo, Wanxia Li Tsai, Massimo Gadina, Mariana J Kaplan, Junya Toguchida, Christian T Mayer, Sergio D Rosenzweig, Luigi D Notarangelo, Kazuhiro Iwai, John Silke, Pamela L Schwartzberg, Bertrand Boisson, Jean-Laurent Casanova, Seiamak Bahram, Anand Prahalad Rao, Nieves Peltzer, Henning Walczak, Najoua Lalaoui, Ivona Aksentijevich, Daniel L Kastner.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.

DOI: https://doi.org/10.1038/s41590-024-01817-w
Cell Rep. 2024 Apr 06. pii: S2211-1247(24)00398-X. [Epub ahead of print]43(4): 114070

NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation.

Tao Yu, Dan Hou, Jiaqi Zhao, Xuan Lu, Wendy K Greentree, Qian Zhao, Min Yang, Don-Gerard Conde, Maurine E Linder, Hening Lin.

  Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation in vivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.

Keywords:  CP: Immunology; NLRP3; ZDHHC7; endotoxic shock; inflammasome; palmitoylation; peritonitis; trans-Golgi network localization

DOI:  https://doi.org/10.1016/j.celrep.2024.114070
Cancer Lett. 2024 Apr 04. pii: S0304-3835(24)00239-8. [Epub ahead of print] 216846

XBP1s activates METTL3/METTL14 for ER-phagy and paclitaxel sensitivity regulation in breast cancer.

Jiajia Wang, Pengyu Fan, Peng Shen, Cong Fan, Pan Zhao, Yao Shen, Kewei Dong, Rui Ling, Suning Chen, Jian Zhang.

  Cancer cells employ the unfolded protein response (UPR) or induce autophagy, especially selective removal of certain ER domains via reticulophagy (termed ER-phagy), to mitigate endoplasmic reticulum (ER) stress for ER homeostasis when encountering microenvironmental stress. N6-methyladenosine (m6A) is one of the most abundant epitranscriptional modifications and plays important roles in various biological processes. However, the molecular mechanism of m6A modification in the ER stress response is poorly understood. In this study, we first found that ER stress could dramatically elevate m6A methylation levels through XBP1s-dependent transcriptional upregulation of METTL3/METTL14 in breast cancer (BC) cells. Further MeRIP sequencing and relevant validation results confirmed that ER stress caused m6A methylation enrichment on target genes for ER-phagy. Mechanistically, METTL3/METTL14 increased ER-phagy machinery formation by promoting m6A modification of the ER-phagy regulators CALCOCO1 and p62, thus enhancing their mRNA stability. Of note, we further confirmed that the chemotherapeutic drug paclitaxel (PTX) could induce ER stress and increase m6A methylation for ER-phagy. Furthermore, the combination of METTL3/METTL14 inhibitors with PTX demonstrated a significant synergistic therapeutic effect in both BC cells and xenograft mice. Thus, our data built a novel bridge on the crosstalk between ER stress, m6A methylation and ER-phagy. Most importantly, our work provides novel evidence of METTL3 and METTL14 as potential therapeutic targets for PTX sensitization in breast cancer.

Keywords:  CALCOCO1; ER stress; ER-Phagy; Paclitaxel; XBP1; m6A; p62

DOI:  https://doi.org/10.1016/j.canlet.2024.216846
Annu Rev Biochem. 2024 Apr 09.

The Endo-Lysosomal Damage Response.

Hemmo Meyer, Bojana Kravic.

Lysosomes are the degradative endpoints of material delivered by endocytosis and autophagy and are therefore particularly prone to damage. Membrane permeabilization or full rupture of lysosomal or late endosomal compartments is highly deleterious because it threatens cellular homeostasis and can elicit cell death and inflammatory signaling. Cells have developed a complex response to endo-lysosomal damage that largely consists of three branches. Initially, a number of repair pathways are activated to restore the integrity of the lysosomal membrane. If repair fails or if damage is too extensive, lysosomes are isolated and degraded by a form of selective autophagy termed lysophagy. Meanwhile, an mTORC1-governed signaling cascade drives biogenesis and regeneration of new lysosomal components to reestablish the full lysosomal capacity of the cell. This damage response is vital to counteract the effects of various conditions, including neurodegeneration and infection, and can constitute a critical vulnerability in cancer cells.

DOI: https://doi.org/10.1146/annurev-biochem-030222-102505
Nat Neurosci. 2024 Apr 08.

Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding.

Ezgi Hacisuleyman, Caryn R Hale, Natalie Noble, Ji-Dung Luo, John J Fak, Misa Saito, Jin Chen, Jonathan S Weissman, Robert B Darnell.

Learning and memory require activity-induced changes in dendritic translation, but which mRNAs are involved and how they are regulated are unclear. In this study, to monitor how depolarization impacts local dendritic biology, we employed a dendritically targeted proximity labeling approach followed by crosslinking immunoprecipitation, ribosome profiling and mass spectrometry. Depolarization of primary cortical neurons with KCl or the glutamate agonist DHPG caused rapid reprogramming of dendritic protein expression, where changes in dendritic mRNAs and proteins are weakly correlated. For a subset of pre-localized messages, depolarization increased the translation of upstream open reading frames (uORFs) and their downstream coding sequences, enabling localized production of proteins involved in long-term potentiation, cell signaling and energy metabolism. This activity-dependent translation was accompanied by the phosphorylation and recruitment of the non-canonical translation initiation factor eIF4G2, and the translated uORFs were sufficient to confer depolarization-induced, eIF4G2-dependent translational control. These studies uncovered an unanticipated mechanism by which activity-dependent uORF translational control by eIF4G2 couples activity to local dendritic remodeling.

DOI: https://doi.org/10.1038/s41593-024-01615-5
Mol Cell. 2024 Apr 02. pii: S1097-2765(24)00225-9. [Epub ahead of print]

An RNA-dependent and phase-separated active subnuclear compartment safeguards repressive chromatin domains.

Luigi Lerra, Martina Panatta, Dominik Bär, Isabella Zanini, Jennifer Yihong Tan, Agnese Pisano, Chiara Mungo, Célia Baroux, Vikram Govind Panse, Ana C Marques, Raffaella Santoro.

  The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.

Keywords:  BAZ2A; H3K27me3; Malat1; RNA; chromatin; ground-state pluripotency; nuclear condensates; nuclear speckles; phase separation

DOI:  https://doi.org/10.1016/j.molcel.2024.03.015
Nat Commun. 2024 Apr 11. 15(1): 3129

Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

Keyu Lv, Shuai Chen, Xulin Xu, Joyce Chiu, Haoqing J Wang, Yunyun Han, Xiaodan Yang, Sheryl R Bowley, Hao Wang, Zhaoming Tang, Ning Tang, Aizhen Yang, Shuofei Yang, Jinyu Wang, Si Jin, Yi Wu, Alvin H Schmaier, Lining A Ju, Philip J Hogg, Chao Fang.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg-/-) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12-/-) and HRG deficiency (by siRNA or Hrg-/-), there is further thrombosis reduction compared to F12-/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.

DOI: https://doi.org/10.1038/s41467-024-47493-0
Nat Commun. 2024 Apr 08. 15(1): 2997

A chemical proteomics approach for global mapping of functional lysines on cell surface of living cell.

Ting Wang, Shiyun Ma, Guanghui Ji, Guoli Wang, Yang Liu, Lei Zhang, Ying Zhang, Haojie Lu.

Cell surface proteins are responsible for many crucial physiological roles, and they are also the major category of drug targets as the majority of therapeutics target membrane proteins on the surface of cells to alter cellular signaling. Despite its great significance, ligand discovery against membrane proteins has posed a great challenge mainly due to the special property of their natural habitat. Here, we design a new chemical proteomic probe OPA-S-S-alkyne that can efficiently and selectively target the lysines exposed on the cell surface and develop a chemical proteomics strategy for global analysis of surface functionality (GASF) in living cells. In total, we quantified 2639 cell surface lysines in Hela cell and several hundred residues with high reactivity were discovered, which represents the largest dataset of surface functional lysine sites to date. We discovered and validated that hyper-reactive lysine residues K382 on tyrosine kinase-like orphan receptor 2 (ROR2) and K285 on Endoglin (ENG/CD105) are at the protein interaction interface in co-crystal structures of protein complexes, emphasizing the broad potential functional consequences of cell surface lysines and GASF strategy is highly desirable for discovering new active and ligandable sites that can be functionally interrogated for drug discovery.

DOI: https://doi.org/10.1038/s41467-024-47033-w
NAR Cancer. 2024 Jun;6(2): zcae015

The DNA Damage Response (DDR) landscape of endometrial cancer defines discrete disease subtypes and reveals therapeutic opportunities.

Xingyuan Zhang, Sayali Joseph, Di Wu, Jessica L Bowser, Cyrus Vaziri.

Genome maintenance is an enabling characteristic that allows neoplastic cells to tolerate the inherent stresses of tumorigenesis and evade therapy-induced genotoxicity. Neoplastic cells also deploy many mis-expressed germ cell proteins termed Cancer Testes Antigens (CTAs) to promote genome maintenance and survival. Here, we present the first comprehensive characterization of the DNA Damage Response (DDR) and CTA transcriptional landscapes of endometrial cancer in relation to conventional histological and molecular subtypes. We show endometrial serous carcinoma (ESC), an aggressive endometrial cancer subtype, is defined by gene expression signatures comprising members of the Replication Fork Protection Complex (RFPC) and Fanconi Anemia (FA) pathway and CTAs with mitotic functions. DDR and CTA-based profiling also defines a subset of highly aggressive endometrioid endometrial carcinomas (EEC) with poor clinical outcomes that share similar profiles to ESC yet have distinct characteristics based on conventional histological and genomic features. Using an unbiased CRISPR-based genetic screen and a candidate gene approach, we confirm that DDR and CTA genes that constitute the ESC and related EEC gene signatures are required for proliferation and therapy-resistance of cultured endometrial cancer cells. Our study validates the use of DDR and CTA-based tumor classifiers and reveals new vulnerabilities of aggressive endometrial cancer where none currently exist.

DOI: https://doi.org/10.1093/narcan/zcae015
Proc Natl Acad Sci U S A. 2024 Apr 16. 121(16): e2315958121

Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

Yen-Ching Chang, Yuan Gao, Joo Yeun Lee, Yi-Jheng Peng, Jennifer Langen, Karen T Chang.

  The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Keywords:  Drosophila; autophagy; neuromuscular junction; synaptic plasticity; synaptic remodeling

DOI:  https://doi.org/10.1073/pnas.2315958121
EMBO Rep. 2024 Apr 11.

Phosphorylation of ELYS promotes its interaction with VAPB at decondensing chromosomes during mitosis.

Christina James, Ulrike Möller, Christiane Spillner, Sabine König, Olexandr Dybkov, Henning Urlaub, Christof Lenz, Ralph H Kehlenbach.

  ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.

Keywords:  ELYS; FFAT-motif; Mitosis; Nuclear Envelope; VAPB

DOI:  https://doi.org/10.1038/s44319-024-00125-6
Sci Adv. 2024 Apr 12. 10(15): eadf7001

The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

Melad Henis, Tabitha Rücker, Robin Scharrenberg, Melanie Richter, Lucas Baltussen, Shuai Hong, Durga Praveen Meka, Birgit Schwanke, Nagammal Neelagandan, Danie Daaboul, Nadeem Murtaza, Christoph Krisp, Sönke Harder, Hartmut Schlüter, Matthias Kneussel, Irm Hermans-Borgmeyer, Joris de Wit, Karun K Singh, Kent E Duncan, Froylan Calderón de Anda.

Genes implicated in translation control have been associated with autism spectrum disorders (ASDs). However, some important genetic causes of autism, including the 16p11.2 microdeletion, bear no obvious connection to translation. Here, we use proteomics, genetics, and translation assays in cultured cells and mouse brain to reveal altered translation mediated by loss of the kinase TAOK2 in 16p11.2 deletion models. We show that TAOK2 associates with the translational machinery and functions as a translational brake by phosphorylating eukaryotic elongation factor 2 (eEF2). Previously, all signal-mediated regulation of translation elongation via eEF2 phosphorylation was believed to be mediated by a single kinase, eEF2K. However, we show that TAOK2 can directly phosphorylate eEF2 on the same regulatory site, but functions independently of eEF2K signaling. Collectively, our results reveal an eEF2K-independent signaling pathway for control of translation elongation and suggest altered translation as a molecular component in the etiology of some forms of ASD.

DOI: https://doi.org/10.1126/sciadv.adf7001
Cancer Res. 2024 Apr 09. OF1-OF15

MILIP Binding to tRNAs Promotes Protein Synthesis to Drive Triple-Negative Breast Cancer.

Si Min Zheng, Yu Chen Feng, Qin Zhu, Ruo Qi Li, Qian Qian Yan, Liu Teng, Yi Meng Yue, Man Man Han, Kaihong Ye, Sheng Nan Zhang, Teng Fei Qi, Cai Xia Tang, Xiao Hong Zhao, Yuan Yuan Zhang, Liang Xu, Ran Xu, Jun Xing, Mark Baker, Tao Liu, Rick F Thorne, Lei Jin, Thomas Preiss, Xu Dong Zhang, Shundong Cang, Jin Nan Gao.

Patients with triple-negative breast cancer (TNBC) have a poor prognosis due to the lack of effective molecular targets for therapeutic intervention. Here we found that the long noncoding RNA (lncRNA) MILIP supports TNBC cell survival, proliferation, and tumorigenicity by complexing with transfer RNAs (tRNA) to promote protein production, thus representing a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, and MILIP silencing suppressed TNBC cell viability and xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its established role in repressing p53 in other types of cancers. Mechanistic investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with the type II tRNAs tRNALeu and tRNASer through their variable loops, which facilitated the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein synthesis and cell viability. Targeting MILIP inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identify MILIP as an RNA translation elongation factor that promotes protein production in TNBC cells and reveal the therapeutic potential of targeting MILIP, alone and in combination with other types of protein synthesis inhibitors, for TNBC treatment.
SIGNIFICANCE: LncRNA MILIP plays a key role in supporting protein production in TNBC by forming complexes with tRNAs and eEF1α1, which confers sensitivity to combined MILIP targeting and protein synthesis inhibitors.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-3046
Nat Neurosci. 2024 Apr 10.

Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration.

Raffaella De Pace, Saikat Ghosh, Veronica H Ryan, Mira Sohn, Michal Jarnik, Paniz Rezvan Sangsari, Nicole Y Morgan, Ryan K Dale, Michael E Ward, Juan S Bonifacino.

In neurons, RNA granules are transported along the axon for local translation away from the soma. Recent studies indicate that some of this transport involves hitchhiking of RNA granules on lysosome-related vesicles. In the present study, we leveraged the ability to prevent transport of these vesicles into the axon by knockout of the lysosome-kinesin adaptor BLOC-one-related complex (BORC) to identify a subset of axonal mRNAs that depend on lysosome-related vesicles for transport. We found that BORC knockout causes depletion of a large group of axonal mRNAs mainly encoding ribosomal and mitochondrial/oxidative phosphorylation proteins. This depletion results in mitochondrial defects and eventually leads to axonal degeneration in human induced pluripotent stem cell (iPSC)-derived and mouse neurons. Pathway analyses of the depleted mRNAs revealed a mechanistic connection of BORC deficiency with common neurodegenerative disorders. These results demonstrate that mRNA transport on lysosome-related vesicles is critical for the maintenance of axonal homeostasis and that its failure causes axonal degeneration.

DOI: https://doi.org/10.1038/s41593-024-01619-1