bims-proteo 2024-05-12 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–05–12
38 papers selected by
Eric Chevet, INSERM

USP20 deubiquitinates and stabilizes the reticulophagy receptor RETREG1/FAM134B to drive reticulophagy.
Mammalian IRE1α dynamically and functionally coalesces with stress granules.
HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.
TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2.
A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22.
A dynamic compositional equilibrium governs mRNA recognition by eIF3.
UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.
The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment.
Selective autophagy of the immunoproteasomes suppresses innate inflammation.
Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.
An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.
CYpHER: Catalytic extracellular targeted protein degradation with high potency and durable effect.
Spatial mapping of hepatic ER and mitochondria architecture reveals zonated remodeling in fasting and obesity.
Development of Inhibitors, Probes, and PROTAC Provides a Complete Toolbox to Study PARK7 in the Living Cell.
Giant organelle vesicles to uncover intracellular membrane mechanics and plasticity.
Obesity disrupts the pituitary-hepatic UPR communication leading to NAFLD progression.
Senataxin deficiency disrupts proteostasis through nucleolar ncRNA-driven protein aggregation.
SUMOylated Golgin45 associates with PML-NB to transcriptionally regulate lipid metabolism genes during heat shock stress.
Ubiquitin-mediated regulation of APE2 protein abundance.
A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.
Enigmatic missense mutations can cause disease via creation of de novo nuclear export signals.
Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks.
The IRE1α/XBP1 pathway sustains cytokine responses of group 3 innate lymphoid cells in inflammatory bowel disease.
Structural mechanism of angiogenin activation by the ribosome.
Endocytosis blocks the vesicular secretion of exosome marker proteins.
DNMT1 regulates human erythropoiesis by modulating cell cycle and endoplasmic reticulum stress in a stage-specific manner.
Molecular basis for the interactions of eIF2β with eIF5, eIF2B, and 5MP1 and their regulation by CK2.
Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.
Design principles to tailor Hsp104 therapeutics.
Morphodynamical adaptation of the endolysosomal system to stress.
Ribosome Assembly and Repair.
Rho-associated kinase regulates Langerhans cell morphology and responsiveness to tissue damage.
Cryo-EM Structure of HRSL Domain Reveals Activating Crossed Helices at the Core of GCN2.
Potential for targeting small heat shock protein modifications.
Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation.
The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.
Decrypting the molecular basis of cellular drug phenotypes by dose-resolved expression proteomics.
Extracellular proteolysis in cancer: Proteases, substrates, and mechanisms in tumor progression and metastasis.

Autophagy. 2024 May 05.

USP20 deubiquitinates and stabilizes the reticulophagy receptor RETREG1/FAM134B to drive reticulophagy.

Man Zhang, Zhangshun Wang, Qing Zhao, Qian Yang, Jieyun Bai, Cuiwei Yang, Zai-Rong Zhang, Yanfen Liu.

  The endoplasmic reticulum (ER) serves as a hub for various essential cellular processes, and maintaining ER homeostasis is essential for cell function. Reticulophagy is a selective process that removes impaired ER subdomains through autophagosome and lysosomal degradation. While the involvement of ubiquitination in autophagy regulation is well-established, its role in reticulophagy remains unclear. In this study, we screened deubiquitinating enzymes (DUBs) involved in reticulophagy and identified USP20 (ubiquitin specific peptidase 20) as a key regulator of reticulophagy under starvation conditions. USP20 specifically cleaves K48- and K63-linked ubiquitin chains on the reticulophagy receptor RETREG1/FAM134B (reticulophagy regulator 1), thereby stabilizing the substrate and promoting reticulophagy. Remarkably, despite lacking a transmembrane domain, USP20 is recruited to the ER through its interaction with VAPs (VAMP associated proteins). VAPs facilitate the recruitment of early autophagy proteins, including WIPI2 (WD repeat domain, phosphoinositide interacting 2), to specific ER subdomains, where USP20 and RETREG1 are enriched. This recruitment of WIPI2 and other proteins plays a crucial role in facilitating RETREG1-mediated reticulophagy in response to nutrient deprivation. These findings highlight the critical role of USP20 in maintaining ER homeostasis by deubiquitinating and stabilizing RETREG1 at distinct ER subdomains, where USP20 further recruits VAPs and promotes efficient reticulophagy.

Keywords:  Autophagy; LC3; Vaps; deubiquitination; endoplasmic reticulum

DOI:  https://doi.org/10.1080/15548627.2024.2347103
Nat Cell Biol. 2024 May 07.

Mammalian IRE1α dynamically and functionally coalesces with stress granules.

Songzi Liu, Xiaoge Zhang, Xin Yao, Guan Wang, Shijia Huang, Peng Chen, Mingliang Tang, Jie Cai, Zhuyin Wu, Yiliang Zhang, Rongzhi Xu, Kai Liu, Kangmin He, Yan Wang, Lei Jiang, Qiong A Wang, Liangyou Rui, Jianmiao Liu, Yong Liu.

Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α-SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α-XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α-SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.

DOI: https://doi.org/10.1038/s41556-024-01418-7
J Cell Biol. 2024 Jul 01. pii: e202308003. [Epub ahead of print]223(7):

HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Yuka Kamada, Yuko Ohnishi, Chikako Nakashima, Aika Fujii, Mana Terakawa, Ikuto Hamano, Uta Nakayamada, Saori Katoh, Noriaki Hirata, Hazuki Tateishi, Ryosuke Fukuda, Hirotaka Takahashi, Gergely L Lukacs, Tsukasa Okiyoneda.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

DOI: https://doi.org/10.1083/jcb.202308003
Cells. 2024 Apr 25. pii: 747. [Epub ahead of print]13(9):

TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2.

Nabil Ahmed, Christian Preisinger, Thomas Wilhelm, Michael Huber.

  The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α-MTDH-VCP complex(es) might enable the treatment of MCL.

Keywords:  ERAD pathway; mastocytosis; metadherin; ubiquitination; valosin-containing protein

DOI:  https://doi.org/10.3390/cells13090747
Nat Commun. 2024 May 06. 15(1): 3789

A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22.

Weize Wang, Ling Liang, Zonglin Dai, Peng Zuo, Shang Yu, Yishuo Lu, Dian Ding, Hongyi Chen, Hui Shan, Yan Jin, Youdong Mao, Yuxin Yin.

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

DOI: https://doi.org/10.1038/s41467-024-48045-2
bioRxiv. 2024 Apr 26. pii: 2024.04.25.581977. [Epub ahead of print]

A dynamic compositional equilibrium governs mRNA recognition by eIF3.

Nicholas A Ide, Riley C Gentry, Margaret A Rudbach, Kyungyoon Yoo, Paloma Kai Velez, Victoria M Comunale, Erik W Hartwick, Colin D Kinz-Thompson, Ruben L Gonzalez, Colin Echeverría Aitken.

Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) in order to drive a diverse set of mechanistic steps during translation. Despite its importance, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is an equilibrium mixture of the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this equilibrium and show that, rather than being driven by the full complex, mRNA binding by eIF3 is instead driven by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in the mRNA recruitment step of translation initiation and establish a mechanistic framework for explaining and investigating the other activities of eIF3.

DOI: https://doi.org/10.1101/2024.04.25.581977
Nucleic Acids Res. 2024 May 06. pii: gkae323. [Epub ahead of print]

UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.

Lukas M Langer, Katharina Kurscheidt, Jérôme Basquin, Fabien Bonneau, Iuliia Iermak, Claire Basquin, Elena Conti.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

DOI: https://doi.org/10.1093/nar/gkae323
Nat Commun. 2024 May 08. 15(1): 3890

The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment.

Wei Xian, Jiaqi Fu, Qinxin Zhang, Chuang Li, Yan-Bo Zhao, Zhiheng Tang, Yi Yuan, Ying Wang, Yan Zhou, Peter S Brzoic, Ning Zheng, Songying Ouyang, Zhao-Qing Luo, Xiaoyun Liu.

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

DOI: https://doi.org/10.1038/s41467-024-48205-4
Autophagy. 2024 May 08.

Selective autophagy of the immunoproteasomes suppresses innate inflammation.

Jiao Zhou, Huihui Li, Kefeng Lu.

  Immunoproteasomes are involved in various inflammatory diseases. Upon stimulation, standard constitutive proteasomes are partially replaced by newly formed immunoproteasomes that promote inflammatory responses. How the upregulated immunoproteasomes are cleared to constrain hyper-inflammation is unknown. Recently, our studies showed that the pan-FGFR inhibitor LY2874455 efficiently activates macroautophagy/autophagy in macrophages, leading to the degradation of the immunoproteasomes. Immunoproteasome subunits are ubiquitinated and recognized by the selective autophagy receptor SQSTM1/p62. LY2874455 suppresses inflammation induced by lipopolysaccharide both in vivo and in vitro through autophagic degradation of the immunoproteasomes. In summary, our work uncovers a mechanism of inflammation suppression by autophagy in macrophages.

Keywords:  Autophagosome; FGFR; immunoproteasome; inflammation; macrophage

DOI:  https://doi.org/10.1080/15548627.2024.2353437
Nat Commun. 2024 May 08. 15(1): 3894

Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.

Linda Lauinger, Anna Andronicos, Karin Flick, Clinton Yu, Geetha Durairaj, Lan Huang, Peter Kaiser.

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

DOI: https://doi.org/10.1038/s41467-024-48184-6
Mol Cell. 2024 Apr 30. pii: S1097-2765(24)00325-3. [Epub ahead of print]

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Jia Ji, Meng-Ke Cui, Rong Zou, Ming-Zhi Wu, Man-Xi Ge, Jiqiang Li, Zai-Rong Zhang.

  Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

Keywords:  ABCG2; ATP13A1; marginally hydrophobic TMD; membrane protein folding; membrane protein topogenesis; multi-spanning membrane protein; poorly hydrophobic TMD; re-orientation; topology; transmembrane domain

DOI:  https://doi.org/10.1016/j.molcel.2024.04.010
bioRxiv. 2024 Apr 23. pii: 2024.02.21.581471. [Epub ahead of print]

CYpHER: Catalytic extracellular targeted protein degradation with high potency and durable effect.

Zachary R Crook, Gregory P Sevilla, Pamela Young, Emily J Girard, Tinh-Doan Phi, Monique Howard, Jason Price, James M Olson, Natalie W Nairn.

Many disease-causing proteins have multiple pathogenic mechanisms, and conventional inhibitors struggle to reliably disrupt more than one. Targeted protein degradation (TPD) can eliminate the protein, and thus all its functions, by directing a cell's protein turnover machinery towards it. Two established strategies either engage catalytic E3 ligases or drive uptake towards the endolysosomal pathway. Here we describe CYpHER ( C atal Y tic pH -dependent E ndolysosomal delivery with R ecycling) technology with potency and durability from a novel catalytic mechanism that shares the specificity and straightforward modular design of endolysosomal uptake. By bestowing pH-dependent release on the target engager and using the rapid-cycling transferrin receptor as the uptake receptor, CYpHER induces endolysosomal target delivery while re-using drug, potentially yielding increased potency and reduced off-target tissue exposure risks. The TfR-based approach allows targeting to tumors that overexpress this receptor and offers the potential for transport to the CNS. CYpHER function was demonstrated in vitro with EGFR and PD-L1, and in vivo with EGFR in a model of EGFR-driven non-small cell lung cancer.

DOI: https://doi.org/10.1101/2024.02.21.581471
Nat Commun. 2024 May 10. 15(1): 3982

Spatial mapping of hepatic ER and mitochondria architecture reveals zonated remodeling in fasting and obesity.

Güneş Parlakgül, Song Pang, Leonardo L Artico, Nina Min, Erika Cagampan, Reyna Villa, Renata L S Goncalves, Grace Yankun Lee, C Shan Xu, Gökhan S Hotamışlıgil, Ana Paula Arruda.

The hepatocytes within the liver present an immense capacity to adapt to changes in nutrient availability. Here, by using high resolution volume electron microscopy, we map how hepatic subcellular spatial organization is regulated during nutritional fluctuations and as a function of liver zonation. We identify that fasting leads to remodeling of endoplasmic reticulum (ER) architecture in hepatocytes, characterized by the induction of single rough ER sheet around the mitochondria, which becomes larger and flatter. These alterations are enriched in periportal and mid-lobular hepatocytes but not in pericentral hepatocytes. Gain- and loss-of-function in vivo models demonstrate that the Ribosome receptor binding protein1 (RRBP1) is required to enable fasting-induced ER sheet-mitochondria interactions and to regulate hepatic fatty acid oxidation. Endogenous RRBP1 is enriched around periportal and mid-lobular regions of the liver. In obesity, ER-mitochondria interactions are distinct and fasting fails to induce rough ER sheet-mitochondrion interactions. These findings illustrate the importance of a regulated molecular architecture for hepatocyte metabolic flexibility.

DOI: https://doi.org/10.1038/s41467-024-48272-7
J Med Chem. 2024 May 07.

Development of Inhibitors, Probes, and PROTAC Provides a Complete Toolbox to Study PARK7 in the Living Cell.

Yuqing Jia, Merve Oyken, Robbert Q Kim, Rayman T N Tjokrodirijo, Arnoud H de Ru, Antonius P A Janssen, Stephan M Hacker, Peter A van Veelen, Paul P Geurink, Aysegul Sapmaz.

The integration of diverse chemical tools like small-molecule inhibitors, activity-based probes (ABPs), and proteolysis targeting chimeras (PROTACs) advances clinical drug discovery and facilitates the exploration of various biological facets of targeted proteins. Here, we report the development of such a chemical toolbox for the human Parkinson disease protein 7 (PARK7/DJ-1) implicated in Parkinson's disease and cancers. By combining structure-guided design, miniaturized library synthesis, and high-throughput screening, we identified two potent compounds, JYQ-164 and JYQ-173, inhibiting PARK7 in vitro and in cells by covalently and selectively targeting its critical residue, Cys106. Leveraging JYQ-173, we further developed a cell-permeable Bodipy probe, JYQ-196, for covalent labeling of PARK7 in living cells and a first-in-class PARK7 degrader JYQ-194 that selectively induces its proteasomal degradation in human cells. Our study provides a valuable toolbox to enhance the understanding of PARK7 biology in cellular contexts and opens new opportunities for therapeutic interventions.

DOI: https://doi.org/10.1021/acs.jmedchem.3c02410
Nat Commun. 2024 May 04. 15(1): 3767

Giant organelle vesicles to uncover intracellular membrane mechanics and plasticity.

Alexandre Santinho, Maxime Carpentier, Julio Lopes Sampaio, Mohyeddine Omrane, Abdou Rachid Thiam.

Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells to a hypotonic medium followed by plasma membrane breakage. By this means, GOVs ranging from 3 to over 10 µm become available for micromanipulation. GOVs are made from organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria, or in contact with one another such as giant mitochondria-associated ER membrane vesicles. We measure the mechanical properties of each organelle-derived GOV and find that they have distinct properties. In GOVs procured from Cos7 cells, for example, bending rigidities tend to increase from the endoplasmic reticulum to the plasma membrane. We also found that the mechanical properties of giant endoplasmic reticulum vesicles (GERVs) vary depending on their interactions with other organelles or the metabolic state of the cell. Lastly, we demonstrate GERVs' biochemical activity through their capacity to synthesize triglycerides and assemble lipid droplets. These findings underscore the potential of GOVs as valuable tools for studying the biophysics and biology of organelles.

DOI: https://doi.org/10.1038/s41467-024-48086-7
Cell Metab. 2024 May 03. pii: S1550-4131(24)00134-7. [Epub ahead of print]

Obesity disrupts the pituitary-hepatic UPR communication leading to NAFLD progression.

Qingwen Qian, Mark Li, Zeyuan Zhang, Shannon W Davis, Kamal Rahmouni, Andrew W Norris, Huojun Cao, Wen-Xing Ding, Gökhan S Hotamisligil, Ling Yang.

  Obesity alters levels of pituitary hormones that govern hepatic immune-metabolic homeostasis, dysregulation of which leads to nonalcoholic fatty liver disease (NAFLD). However, the impact of obesity on intra-pituitary homeostasis is largely unknown. Here, we uncovered a blunted unfolded protein response (UPR) but elevated inflammatory signatures in pituitary glands of obese mice and humans. Furthermore, we found that obesity inflames the pituitary gland, leading to impaired pituitary inositol-requiring enzyme 1α (IRE1α)-X-box-binding protein 1 (XBP1) UPR branch, which is essential for protecting against pituitary endocrine defects and NAFLD progression. Intriguingly, pituitary IRE1-deletion resulted in hypothyroidism and suppressed the thyroid hormone receptor B (THRB)-mediated activation of Xbp1 in the liver. Conversely, activation of the hepatic THRB-XBP1 axis improved NAFLD in mice with pituitary UPR defect. Our study provides the first evidence and mechanism of obesity-induced intra-pituitary cellular defects and the pathophysiological role of pituitary-liver UPR communication in NAFLD progression.

Keywords:  IRE1; NAFLD; UPR; obesity; pituitary gland; thyroid hormone

DOI:  https://doi.org/10.1016/j.cmet.2024.04.014
J Cell Biol. 2024 Jul 01. pii: e202309036. [Epub ahead of print]223(7):

Senataxin deficiency disrupts proteostasis through nucleolar ncRNA-driven protein aggregation.

Xuemei Wen, Hengyi Xu, Phillip R Woolley, Olivia M Conway, Jun Yao, Andreas Matouschek, Alan M Lambowitz, Tanya T Paull.

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.

DOI: https://doi.org/10.1083/jcb.202309036
Commun Biol. 2024 May 06. 7(1): 532

SUMOylated Golgin45 associates with PML-NB to transcriptionally regulate lipid metabolism genes during heat shock stress.

Shuaiyang Jing, Jingkai Gao, Neeraj Tiwari, Yulei Du, Lianhui Zhu, Bopil Gim, Yi Qian, Xihua Yue, Intaek Lee.

Golgin tethers are known to mediate vesicular transport in the secretory pathway, whereas it is relatively unknown whether they may mediate cellular stress response within the cell. Here, we describe a cellular stress response during heat shock stress via SUMOylation of a Golgin tether, Golgin45. We found that Golgin45 is a SUMOylated Golgin via SUMO1 under steady state condition. Upon heat shock stress, the Golgin enters the nucleus by interacting with Importin-β2 and gets further modified by SUMO3. Importantly, SUMOylated Golgin45 appears to interact with PML and SUMO-deficient Golgin45 mutant functions as a dominant negative for PML-NB formation during heat shock stress, suppressing transcription of lipid metabolism genes. These results indicate that Golgin45 may play a role in heat stress response by transcriptional regulation of lipid metabolism genes in SUMOylation-dependent fashion.

DOI: https://doi.org/10.1038/s42003-024-06232-3
J Biol Chem. 2024 May 03. pii: S0021-9258(24)01838-6. [Epub ahead of print] 107337

Ubiquitin-mediated regulation of APE2 protein abundance.

Anne McMahon, Jianjun Zhao, Shan Yan.

  APE2 plays important roles in the maintenance of genomic and epigenomic stability including DNA repair and DNA damage response. Accumulating evidence has suggested that APE2 is upregulated in multiple cancers at the protein and mRNA levels, and that APE2 upregulation is correlative with higher and lower overall survival of cancer patients depending on tumor type. However, it remains unknown how APE2 protein abundance is maintained and regulated in cells. Here, we provide the first evidence of APE2 regulation via the post-translational modification (PTM) Ubiquitin. APE2 is poly-ubiquitinated via K48-linked chains and degraded via the Ubiquitin-Proteasome system where K371 is the key residue within APE2 responsible for its ubiquitination and degradation. We further characterize MKRN3 as the E3 ubiquitin ligase for APE2 ubiquitination in cells and in vitro. In summary, this study offers the first definition of the APE2 proteostasis network and lays the foundation for future studies pertaining to the PTM regulation and functions of APE2 in genome integrity and cancer etiology/treatment.

Keywords:  APE2; DNA damage response; MKRN3; Post-translational modification (PTM); Proteostasis; Ubiquitin

DOI:  https://doi.org/10.1016/j.jbc.2024.107337
J Cell Biol. 2024 Jul 01. pii: e202309015. [Epub ahead of print]223(7):

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Dan A Tudorica, Bishal Basak, Alexia S Puerta Cordova, Grace Khuu, Kevin Rose, Michael Lazarou, Erika L F Holzbaur, James H Hurley.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

DOI: https://doi.org/10.1083/jcb.202309015
bioRxiv. 2024 Apr 24. pii: 2024.04.24.590854. [Epub ahead of print]

Enigmatic missense mutations can cause disease via creation of de novo nuclear export signals.

By Michael McConville, Toby Thomas, Ryan Beckner, Catherine Valadez, YuhMin Chook, Stephen Chung, Glen Liszczak.

Disease-causing missense mutations that occur within structurally and functionally unannotated protein regions can guide researchers to new mechanisms of protein regulation and dysfunction. Here, we report that the thrombocytopenia-, myelodysplastic syndromes-, and leukemia-associated P214L mutation in the transcriptional regulator ETV6 creates an XPO1-dependent nuclear export signal to cause protein mislocalization. Strategies to disrupt XPO1 activity fully restore ETV6 P214L protein nuclear localization and transcription regulation activity. Mechanistic insight inspired the design of a 'humanized' ETV6 mice, which we employ to demonstrate that the germline P214L mutation is sufficient to elicit severe defects in thrombopoiesis and hematopoietic stem cell maintenance. Beyond ETV6, we employed computational methods to uncover rare disease-associated missense mutations in unrelated proteins that create a nuclear export signal to disrupt protein function. Thus, missense mutations that operate through this mechanism should be predictable and may suggest rational therapeutic strategies for associated diseases.

DOI: https://doi.org/10.1101/2024.04.24.590854
bioRxiv. 2024 Apr 22. pii: 2024.04.22.590579. [Epub ahead of print]

Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks.

Meiyan Jin, Yuichiro Iwamoto, Cyna Shirazinejad, David G Drubin.

During clathrin-mediated endocytosis (CME), dozens of proteins are recruited to nascent CME sites on the plasma membrane. Coordination of endocytic protein recruitment in time and space is important for efficient CME. Here, we show that the multivalent scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. By live-cell imaging of genome-edited cells, we observed that endogenously labeled ITSN1 is recruited to CME sites shortly after they begin to assemble. Knocking down ITSN1 impaired endocytic protein recruitment during the stabilization stage of CME site assembly. Artificially locating ITSN1 to the mitochondria surface was sufficient to assemble puncta consisting of CME initiation proteins, including EPS15, FCHO, adaptor proteins, the AP2 complex and epsin1 (EPN1), and the vesicle scission GTPase dynamin2 (DNM2). ITSN1 can form puncta and recruit DNM2 independently of EPS15/FCHO or EPN1. Our work redefines ITSN1's primary endocytic role as organizing and stabilizing the CME protein interaction networks rather than a previously suggested role in initiation and provides new insights into the multi-step and multi-zone organization of CME site assembly.

DOI: https://doi.org/10.1101/2024.04.22.590579
J Clin Invest. 2024 May 09. pii: e174198. [Epub ahead of print]

The IRE1α/XBP1 pathway sustains cytokine responses of group 3 innate lymphoid cells in inflammatory bowel disease.

Siyan Cao, Jose Luis Fachi, Kaiming Ma, Alina Ulezko Antonova, Qianli Wang, Zhangying Cai, Randal J Kaufman, Matthew A Ciorba, Parakkal Deepak, Marco Colonna.

  Group 3 innate lymphoid cells (ILC3s) are key players in intestinal homeostasis. Endoplasmic reticulum (ER) stress is linked to inflammatory bowel disease (IBD). Herein, we used cell culture, mouse models, and human specimens to examine if ER stress in ILC3s impacts IBD pathophysiology. We show that mouse intestinal ILC3s exhibited a 24h-rhythmic expression pattern of the master ER stress response regulator, IRE1α/XBP1. Proinflammatory cytokine IL-23 selectively stimulated IRE1α/XBP1 in mouse ILC3s through mitochondrial reactive oxygen species (mtROS). IRE1α/XBP1 was activated in ILC3s of mice exposed to experimental colitis and in inflamed human IBD specimens. Mice with Ire1α deletion in ILC3s (Ire1αΔRorc) showed reduced expression of ER stress response and cytokine genes including Il22 in ILC3s and were highly vulnerable to infections and colitis. Administration of IL-22 counteracted their colitis susceptibility. In human ILC3s, IRE1 inhibitors suppressed cytokine production, which was upregulated by an IRE1 activator. Moreover, the frequencies of intestinal XBP1s+ ILC3s in Crohn's disease patients before administration of ustekinumab, an anti-IL-12/IL-23 antibody, positively correlated with response to treatment. We demonstrate that a non-canonical mtROS-IRE1α/XBP1 pathway augments cytokine production by ILC3s and identify XBP1s+ ILC3s as a potential biomarker for predicting response to anti-IL-23 therapies in IBD.

Keywords:  Cell stress; Gastroenterology; Immunology; Inflammatory bowel disease; Innate immunity

DOI:  https://doi.org/10.1172/JCI174198
Nature. 2024 May 08.

Structural mechanism of angiogenin activation by the ribosome.

Anna B Loveland, Cha San Koh, Robin Ganesan, Allan Jacobson, Andrei A Korostelev.

Angiogenin, an RNase A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases, and epigenetic inheritance 1-10. Upon activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression 11-15. The catalytic activity of isolated angiogenin, however, is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle 3,16-23. Here, we uncover these mechanisms using biochemical assays and cryogenic electron microscopy. Our work reveals that the cytosolic ribosome is the long-sought activator of angiogenin. A 2.8-Å resolution cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic center, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S•angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, whose abundance increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.

DOI: https://doi.org/10.1038/s41586-024-07508-8
Sci Adv. 2024 May 10. 10(19): eadi9156

Endocytosis blocks the vesicular secretion of exosome marker proteins.

Yiwei Ai, Chenxu Guo, Marta Garcia-Contreras, Laura S Sánchez B, Andras Saftics, Oluwapelumi Shodubi, Shankar Raghunandan, Junhao Xu, Shang Jui Tsai, Yi Dong, Rong Li, Tijana Jovanovic-Talisman, Stephen J Gould.

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

DOI: https://doi.org/10.1126/sciadv.adi9156
Cell Death Differ. 2024 May 08.

DNMT1 regulates human erythropoiesis by modulating cell cycle and endoplasmic reticulum stress in a stage-specific manner.

Qianqian Yang, Lixiang Chen, Hengchao Zhang, Mengjia Li, Lei Sun, Xiuyun Wu, Huizhi Zhao, Xiaoli Qu, Xiuli An, Ting Wang.

The dynamic balance of DNA methylation and demethylation is required for erythropoiesis. Our previous transcriptomic analyses revealed that DNA methyltransferase 1 (DNMT1) is abundantly expressed in erythroid cells at all developmental stages. However, the role and molecular mechanisms of DNMT1 in human erythropoiesis remain unknown. Here we found that DNMT1 deficiency led to cell cycle arrest of erythroid progenitors which was partially rescued by treatment with a p21 inhibitor UC2288. Mechanically, this is due to decreased DNA methylation of p21 promoter, leading to upregulation of p21 expression. In contrast, DNMT1 deficiency led to increased apoptosis during terminal stage by inducing endoplasmic reticulum (ER) stress in a p21 independent manner. ER stress was attributed to the upregulation of RPL15 expression due to the decreased DNA methylation at RPL15 promoter. The upregulated RPL15 expression subsequently caused a significant upregulation of core ribosomal proteins (RPs) and thus ultimately activated all branches of unfolded protein response (UPR) leading to the excessive ER stress, suggesting a role of DNMT1 in maintaining protein homeostasis during terminal erythroid differentiation. Furthermore, the increased apoptosis was significantly rescued by the treatment of ER stress inhibitor TUDCA. Our findings demonstrate the stage-specific role of DNMT1 in regulating human erythropoiesis and provide new insights into regulation of human erythropoiesis.

DOI: https://doi.org/10.1038/s41418-024-01305-6
bioRxiv. 2024 Apr 25. pii: 2024.04.25.591181. [Epub ahead of print]

Molecular basis for the interactions of eIF2β with eIF5, eIF2B, and 5MP1 and their regulation by CK2.

Paul A Wagner, Meimei Song, Ralf Ficner, Bernhard Kuhle, Assen Marintchev.

The heterotrimeric GTPase eukaryotic translation initiation factor 2 (eIF2) delivers the initiator Met-tRNAi to the ribosomal translation preinitiation complex. eIF2β has three lysine-rich repeats (K-boxes) in its N-terminal tail, which are important for binding to the GTPase-activating protein (GAP) eIF5, the guanine nucleotide exchange factor (GEF) eIF2B, and the regulator eIF5-mimic protein (5MP). Here, we combine X-ray crystallography with NMR to understand the molecular basis and dynamics of these interactions. The crystal structure of yeast eIF5-CTD in complex with K-box 3 of eIF2β reveals an extended binding site on eIF2β, far beyond the K-box. We show that human eIF5, eIF2Bε, and 5MP1 can all bind to each of the three K-boxes, while reducing each other's affinities. Moreover, all these affinities are increased by CK2 phosphomimetic mutations. Our results reveal how eIF5, eIF2B, and 5MP displace each other from eIF2, and elucidate the role of CK2 in remodeling the translation apparatus.

DOI: https://doi.org/10.1101/2024.04.25.591181
Commun Biol. 2024 May 09. 7(1): 554

Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.

Sanghee Shin, Song-Yi Lee, Myeong-Gyun Kang, Dong-Gi Jang, Jeesoo Kim, Hyun-Woo Rhee, Jong-Seo Kim.

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.

DOI: https://doi.org/10.1038/s42003-024-06112-w
bioRxiv. 2024 Apr 28. pii: 2024.04.26.591398. [Epub ahead of print]

Design principles to tailor Hsp104 therapeutics.

JiaBei Lin, Peter J Carman, Craig W Gambogi, Nathan M Kendsersky, Edward Chuang, Stephanie N Gates, Adam L Yokom, Alexandrea N Rizo, Daniel R Southworth, James Shorter.

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off- target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics.

DOI: https://doi.org/10.1101/2024.04.26.591398
FEBS J. 2024 May 05.

Morphodynamical adaptation of the endolysosomal system to stress.

Juliane Da Graça, Cédric Delevoye, Etienne Morel.

  In eukaryotes, the spatiotemporal control of endolysosomal organelles is central to the maintenance of homeostasis. By providing an interface between the cytoplasm and external environment, the endolysosomal system is placed at the forefront of the response to a wide range of stresses faced by cells. Endosomes are equipped with a dedicated set of membrane-associated proteins that ensure endosomal functions as well as crosstalk with the secretory or the autophagy pathways. Morphodynamical processes operate through local spatialization of subdomains, enabling specific remodeling and membrane contact capabilities. Consequently, the plasticity of endolysosomal organelles can be considered a robust and flexible tool exploited by cells to cope with homeostatic deviations. In this review, we provide insights into how the cellular responses to various stresses (osmotic, UV, nutrient deprivation, or pathogen infections) rely on the adaptation of the endolysosomal system morphodynamics.

Keywords:  autophagy; endocytic pathway; endolysosomes; endosomes; lysosome‐related organelles; membrane dynamics and contact sites; organelles; pathogen infection; plasma membrane; stress response

DOI:  https://doi.org/10.1111/febs.17154
Annu Rev Cell Dev Biol. 2024 May 09.

Ribosome Assembly and Repair.

Yoon-Mo Yang, Katrin Karbstein.

Ribosomes synthesize protein in all cells. Maintaining both the correct number and composition of ribosomes is critical for protein homeostasis. To address this challenge, cells have evolved intricate quality control mechanisms during assembly to ensure that only correctly matured ribosomes are released into the translating pool. However, these assembly-associated quality control mechanisms do not deal with damage that arises during the ribosomes' exceptionally long lifetimes and might equally compromise their function or lead to reduced ribosome numbers. Recent research has revealed that ribosomes with damaged ribosomal proteins can be repaired by the release of the damaged protein, thereby ensuring ribosome integrity at a fraction of the energetic cost of producing new ribosomes, appropriate for stress conditions. In this article, we cover the types of ribosome damage known so far, and then we review the known repair mechanisms before surveying the literature for possible additional instances of repair.

DOI: https://doi.org/10.1146/annurev-cellbio-111822-113326
Cell Rep. 2024 May 09. pii: S2211-1247(24)00536-9. [Epub ahead of print]43(5): 114208

Rho-associated kinase regulates Langerhans cell morphology and responsiveness to tissue damage.

Eric Peterman, Elgene J A Quitevis, Camille E A Goo, Jeffrey P Rasmussen.

  Skin damage requires efficient immune cell responses to restore organ function. Epidermal-resident immune cells known as Langerhans cells use dendritic protrusions to surveil the skin microenvironment, which contains keratinocytes and peripheral axons. The mechanisms governing Langerhans cell dendrite dynamics and responses to tissue damage are poorly understood. Using skin explants from adult zebrafish, we show that Langerhans cells maintain normal surveillance following axonal degeneration and use their dendrites to engulf small axonal debris. By contrast, a ramified-to-rounded shape transition accommodates the engulfment of larger keratinocyte debris. We find that Langerhans cell dendrites are populated with actin and sensitive to a broad-spectrum actin inhibitor. We show that Rho-associated kinase (ROCK) inhibition leads to elongated dendrites, perturbed clearance of large debris, and reduced Langerhans cell migration to epidermal wounds. Our work describes the dynamics of Langerhans cells and involvement of the ROCK pathway in immune cell responses.

Keywords:  CP: Cell biology; ROCK; actin cytoskeleton; phagocytosis; tissue-resident macrophage; zebrafish

DOI:  https://doi.org/10.1016/j.celrep.2024.114208
bioRxiv. 2024 Apr 25. pii: 2024.04.24.591037. [Epub ahead of print]

Cryo-EM Structure of HRSL Domain Reveals Activating Crossed Helices at the Core of GCN2.

Kristina Solorio-Kirpichyan, Xiao Fan, Dmitrij Golovenko, Andrei A Korostelev, Nieng Yan, Alexei Korennykh.

GCN2 is a conserved receptor kinase activating the Integrated Stress Response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a h istidyl t R NA s ynthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain. How HRSL contributes to GCN2 signaling remains unknown. Here we report a 3.2 Å cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus . This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and kinase domains, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. Remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This autoinhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor.
Significance statement: Regulation of protein synthesis (translation) is a central mechanism by which eukaryotic cells adapt to stressful conditions. In starving cells, this translational adaptation is achieved via the receptor kinase GCN2, which stays inactive under normal conditions, but is switched on under stress. The molecular mechanism of GCN2 switching is not well understood due to the presence of a structurally and biochemically uncharacterized h istidyl t R NA s ynthetase-like domain (HRSL) at the core of GCN2. Here we use single-particle cryo-EM and biochemistry to elucidate the structure and function of HRSL. We identify a structure at the kinase/HRSL interface, which forms crossed helices and helps position GCN2 kinase domains for activation. These data clarify the molecular mechanism of GCN2 regulation.

DOI: https://doi.org/10.1101/2024.04.24.591037
Trends Pharmacol Sci. 2024 May 03. pii: S0165-6147(24)00067-1. [Epub ahead of print]

Potential for targeting small heat shock protein modifications.

Binyou Wang, Matthew R Pratt.

  Small heat shock proteins (sHSPs) play key roles in cellular stress and several human diseases. The direct effects of some post-translational modifications (PTMs) on certain sHSPs have been characterized, raising the possibility that small molecules could be used to modulate these modifications and indirectly up- or downregulate sHSP activity.

Keywords:  O-GlcNAc; glycation; heat shock proteins; phosphorylation; post-translational modifications; protein aggregation

DOI:  https://doi.org/10.1016/j.tips.2024.04.002
bioRxiv. 2024 Apr 24. pii: 2024.04.24.590974. [Epub ahead of print]

Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation.

Yingjie Hu, Victor A Lopez, Hengyi Xu, James P Pfister, Bing Song, Kelly A Servage, Masahiro Sakurai, Benjamin T Jones, Joshua T Mendell, Tao Wang, Jun Wu, Alan M Lambowitz, Diana R Tomchick, Krzysztof Pawłowski, Vincent S Tagliabracci.

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here we identify C12orf29 as an atypical ATP-grasp enzyme that ligates RNA. Human C12orf29 and its homologs auto-adenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. C12orf29 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro . Genetic depletion of c12orf29 in female mice alters global tRNA levels in brain. Furthermore, crystal structures of a C12orf29 homolog from Yasminevirus bound to nucleotides reveal a minimal and atypical RNA ligase fold with a unique active site architecture that participates in catalysis. Collectively, our results identify C12orf29 as an RNA ligase and suggest its involvement in tRNA biology.
Significance Statement: ATP-grasp enzymes share an atypical ATP-binding fold and catalyze a diverse set of reactions involved in many essential cellular processes. We identified C12orf29 as an atypical ATP-grasp enzyme. Our biochemical and structural characterizations reveal this enzyme to be a 5' to 3' RNA ligase, structurally and functionally similar to the phage T4 RNA ligase. C12orf29 can ligate tRNAs in vitro and C12orf29 knockout female mice have altered tRNA levels in brain. We also report structures of C12orf29, which have revealed critical insights into the mode of ATP binding and catalysis. Our work suggests that C12orf29 may be a new player in the regulation of tRNAs.

DOI: https://doi.org/10.1101/2024.04.24.590974
Mol Cell. 2024 Apr 26. pii: S1097-2765(24)00285-5. [Epub ahead of print]

The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

Dimitrios Papadopoulos, Stefanie Anh Ha, Daniel Fleischhauer, Leonie Uhl, Timothy J Russell, Ivan Mikicic, Katharina Schneider, Annika Brem, Omkar Rajendra Valanju, Giacomo Cossa, Peter Gallant, Christina Schuelein-Voelk, Hans Michael Maric, Petra Beli, Gabriele Büchel, Seychelle M Vos, Martin Eilers.

  The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.

Keywords:  MAX; MNT; MXD; MYC; MYCN; NEXT; ZCCHC8; exosome; neuroblastoma

DOI:  https://doi.org/10.1016/j.molcel.2024.04.007
Nat Biotechnol. 2024 May 07.

Decrypting the molecular basis of cellular drug phenotypes by dose-resolved expression proteomics.

Stephan Eckert, Nicola Berner, Karl Kramer, Annika Schneider, Julian Müller, Severin Lechner, Sarah Brajkovic, Amirhossein Sakhteman, Christian Graetz, Jonas Fackler, Michael Dudek, Michael W Pfaffl, Percy Knolle, Stephanie Wilhelm, Bernhard Kuster.

Proteomics is making important contributions to drug discovery, from target deconvolution to mechanism of action (MoA) elucidation and the identification of biomarkers of drug response. Here we introduce decryptE, a proteome-wide approach that measures the full dose-response characteristics of drug-induced protein expression changes that informs cellular drug MoA. Assaying 144 clinical drugs and research compounds against 8,000 proteins resulted in more than 1 million dose-response curves that can be interactively explored online in ProteomicsDB and a custom-built Shiny App. Analysis of the collective data provided molecular explanations for known phenotypic drug effects and uncovered new aspects of the MoA of human medicines. We found that histone deacetylase inhibitors potently and strongly down-regulated the T cell receptor complex resulting in impaired human T cell activation in vitro and ex vivo. This offers a rational explanation for the efficacy of histone deacetylase inhibitors in certain lymphomas and autoimmune diseases and explains their poor performance in treating solid tumors.

DOI: https://doi.org/10.1038/s41587-024-02218-y
J Biol Chem. 2024 May 06. pii: S0021-9258(24)01848-9. [Epub ahead of print] 107347

Extracellular proteolysis in cancer: Proteases, substrates, and mechanisms in tumor progression and metastasis.

Evette S Radisky.

  A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.

Keywords:  activity-based protein profiling; aspartic protease; cancer progression; cathepsin; cysteine protease; degradomics; extracellular matrix; metalloprotease; metastasis; protease; protein protease inhibitor; proteolysis; proteolytic signaling; serine protease; tumor microenvironment; zymogen

DOI:  https://doi.org/10.1016/j.jbc.2024.107347