bims-proteo 2022-10-09 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022–10–09
23 papers selected by
Eric Chevet, INSERM

The functional importance of VCP to maintaining cellular protein homeostasis.
The mechano-ubiquitinome of articular cartilage: Differential ubiquitination and activation of a group of ER-associated DUBs and ER stress regulators.
Linear ubiquitination of PTEN impairs its function to promote prostate cancer progression.
Integrated stress response is involved in the 24(S)-hydroxycholesterol-induced unconventional cell death mechanism.
UPRmt and coordinated UPRER in type 2 diabetes.
An emerging role for the endoplasmic reticulum in stress granule biogenesis.
Probing protein ubiquitination in live cells.
A Noncanonical Autophagy Function of ATG9A for Golgi Integrity and Dynamics.
Thermosensation in Caenorhabditis elegans is linked to ubiquitin-dependent protein turnover via insulin and calcineurin signalling.
Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.
Destabilization of TP53 by USP10 is essential for neonatal autophagy and survival.
Model for ring closure in ER tubular network dynamics.
DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on protein substrates.
Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry.
Autophagy induction promoted by m6A reader YTHDF3 through translation upregulation of FOXO3 mRNA.
Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle.
DDX17 is required for efficient DSB repair at DNA:RNA hybrid deficient loci.
Proteotoxic stress-induced apoptosis in cancer cells: understanding the susceptibility and enhancing the potency.
Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance.
Post-translational modifications reshape the antigenic landscape of the MHC I immunopeptidome in tumors.
X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.
Aging compromises human islet beta cell function and identity by decreasing transcription factor activity and inducing ER stress.
Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with challenges to proteostasis.

Biochem Soc Trans. 2022 Oct 05. pii: BST20220648. [Epub ahead of print]

The functional importance of VCP to maintaining cellular protein homeostasis.

Brittany A Ahlstedt, Rakesh Ganji, Malavika Raman.

  The AAA-ATPase (ATPases associated with diverse cellular activities) valosin-containing protein (VCP), is essential for many cellular pathways including but not limited to endoplasmic reticulum-associated degradation (ERAD), DNA damage responses, and cell cycle regulation. VCP primarily identifies ubiquitylated proteins in these pathways and mediates their unfolding and degradation by the 26S proteasome. This review summarizes recent research on VCP that has uncovered surprising new ways that this ATPase is regulated, new aspects of recognition of substrates and novel pathways and substrates that utilize its activity.

Keywords:  autophagy; neurodegeneration; organelles; protein quality control; ubiquitin

DOI:  https://doi.org/10.1042/BST20220648
Mol Cell Proteomics. 2022 Sep 28. pii: S1535-9476(22)00227-4. [Epub ahead of print] 100419

The mechano-ubiquitinome of articular cartilage: Differential ubiquitination and activation of a group of ER-associated DUBs and ER stress regulators.

Nitchakarn Kaokhum, Adán Pinto-Fernández, Mark Wilkinson, Benedikt M Kessler, Heba M Ismail.

Understanding how connective tissue cells respond to mechanical stimulation is important to human health and disease processes in musculoskeletal diseases. Injury to articular cartilage is a key risk factor in predisposition to tissue damage and degenerative osteoarthritis. Recently, we have discovered that mechanical injury to connective tissues including murine and porcine articular cartilage causes a significant increase in Lysine 63- polyubiquitination. Here we identified the ubiquitin signature that is unique to injured articular cartilage tissue post mechanical injury (the "mechano-ubiquitinome"). A total of 463 ubiquitinated peptides were identified, with an enrichment of ubiquitinated peptides of proteins involved in protein processing in the endoplasmic reticulum (ER), also known as the ER-associated degradation (ERAD) response, including YOD1, BRCC3, ATXN3 and USP5 as well as the ER stress regulators, RAD23B, VCP/p97 and Ubiquilin 1. Enrichment of these proteins suggested an injury-induced ER stress response and, for instance, ER stress markers DDIT3/CHOP and BIP/GRP78 were upregulated following cartilage injury on the protein and gene expression levels. Similar ER stress induction was also observed in response to tail fin injury in zebrafish larvae, suggesting a generic response to tissue injury. Furthermore, a rapid increase in global DUB activity following injury and significant activity in human osteoarthritic cartilage was observed using DUB specific activity probes. Combined, these results implicate the involvement of ubiquitination events and activation of a set of DUBs and ER stress regulators in cellular responses to cartilage tissue injury and in osteoarthritic cartilage tissues. This link through the ERAD pathway makes this protein set attractive for further investigation in in vivo models of tissue injury and for targeting in osteoarthritis and related musculoskeletal diseases.

DOI: https://doi.org/10.1016/j.mcpro.2022.100419
Oncogene. 2022 Oct 03.

Linear ubiquitination of PTEN impairs its function to promote prostate cancer progression.

Yanmin Guo, Jianfeng He, Hailong Zhang, Ran Chen, Lian Li, Xiaojia Liu, Caihu Huang, Zhe Qiang, Zihan Zhou, Yanli Wang, Jian Huang, Xian Zhao, Junke Zheng, Guo-Qiang Chen, Jianxiu Yu.

PTEN is frequently mutated in human cancers, which leads to the excessive activation of PI3K/AKT signaling and thus promotes tumorigenesis and drug resistance. Met1-linked ubiquitination (M1-Ubi) is also involved in cancer progression, but the mechanism is poorly defined. Here we find that HOIP, one important component of linear ubiquitin chain assembly complex (LUBAC), promotes prostate cancer (PCa) progression by enhancing AKT signaling in a PTEN-dependent manner. Mechanistically, PTEN is modified by M1-Ubi at two sites K144 and K197, which significantly inhibits PTEN phosphatase activity and thus accelerates PCa progression. More importantly, we identify that the high-frequency mutants PTENR173H and PTENR173C in PCa patients showed the enhanced level of M1-Ubi, which impairs PTEN function in inhibition of AKT phosphorylation and cell growth. We also find that HOIP depletion sensitizes PCa cells to therapeutic agents BKM120 and Enzalutamide. Furthermore, the clinical data analyses confirm that HOIP is upregulated and positively correlated with AKT activation in PCa patient specimen, which may promote PCa progression and increase the risk of PCa biochemical relapse. Together, our study reveals a key role of PTEN M1-Ubi in regulation of AKT activation and PCa progression, which may propose a new strategy for PCa therapy.

DOI: https://doi.org/10.1038/s41388-022-02485-6
Cell Death Discov. 2022 Oct 04. 8(1): 406

Integrated stress response is involved in the 24(S)-hydroxycholesterol-induced unconventional cell death mechanism.

Yasuomi Urano, Shoya Osaki, Ren Chiba, Noriko Noguchi.

Perturbation of proteostasis triggers the adaptive responses that contribute to the homeostatic pro-survival response, whereas disruption of proteostasis can ultimately lead to cell death. Brain-specific oxysterol-i.e., 24(S)-hydroxycholesterol (24S-OHC)-has been shown to cause cytotoxicity when esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the endoplasmic reticulum (ER). Here, we show that the accumulation of 24S-OHC esters caused phosphorylation of eukaryotic translation initiator factor 2α (eIF2α), dissociation of polysomes, and formation of stress granules (SG), resulting in robust downregulation of global protein de novo synthesis in human neuroblastoma SH-SY5Y cells. We also found that integrated stress response (ISR) activation through PERK and GCN2 activation induced by 24S-OHC treatment caused eIF2α phosphorylation. 24S-OHC-inducible SG formation and cell death were suppressed by inhibition of ISR. These results show that ACAT1-mediated 24S-OHC esterification induced ISR and formation of SG, which play crucial roles in 24S-OHC-inducible protein synthesis inhibition and unconventional cell death.

DOI: https://doi.org/10.1038/s41420-022-01197-w
Front Cell Dev Biol. 2022 ;10 974083

UPRmt and coordinated UPRER in type 2 diabetes.

Zhanfang Kang, Feng Chen, Wanhui Wu, Rui Liu, Tianda Chen, Fang Xu.

  The mitochondrial unfolded protein response (UPRmt) is a molecular mechanism that maintains mitochondrial proteostasis under stress and is closely related to various metabolic diseases, such as type 2 diabetes (T2D). Similarly, the unfolded protein response of the endoplasmic reticulum (UPRER) is responsible for maintaining proteomic stability in the endoplasmic reticulum (ER). Since the mitochondria and endoplasmic reticulum are the primary centers of energy metabolism and protein synthesis in cells, respectively, a synergistic mechanism must exist between UPRmt and UPRER to cooperatively resist stresses such as hyperglycemia in T2D. Increasing evidence suggests that the protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) signaling pathway is likely an important node for coordinating UPRmt and UPRER. The PERK pathway is activated in both UPRmt and UPRER, and its downstream molecules perform important functions. In this review, we discuss the mechanisms of UPRmt, UPRER and their crosstalk in T2D.

Keywords:  PERK (PKR-like endoplasmic reticulum kinase); T2D; UPR; UPRmt; mitochondia; unfolded protein response

DOI:  https://doi.org/10.3389/fcell.2022.974083
Semin Cell Dev Biol. 2022 Oct 03. pii: S1084-9521(22)00275-0. [Epub ahead of print]

An emerging role for the endoplasmic reticulum in stress granule biogenesis.

Christopher V Nicchitta.

  Stress granules (SGs), structurally dynamic, optically resolvable, macromolecular assemblies of mRNAs, RNA binding proteins (RBPs), translation factors, ribosomal subunits, as well as other interacting proteins, assemble in response to cell stress conditions that elicit phosphorylation of eukaryotic initiation factor 2α (eIF2α) and consequently, the inactivation of translation initiation. SG biology is conserved throughout eukaryotes and has recently been linked to the pathological sequelae of neurodegenerative disorders, cancer biology, and viral infection. Substantial insights into mechanisms of SG biogenesis, and more broadly the phenomenon of biological liquid-liquid phase separation (LLPS), have been aided by detailed proteomic and transcriptomic studies as well as in vitro reconstitution approaches. A particularly interesting and largely unexplored element of SG biology is the cell biological context of SG biogenesis, including its subcellular organization and more recently, evidence that the endoplasmic reticulum (ER) membrane may serve important functions in RNA granule biology generally and SG biogenesis specifically. A central role for the ER in SG biogenesis is discussed and a hypothesis linking SG formation on the ER to the trafficking, localization and de novo translation of newly exported mRNAs is presented.

Keywords:  Endoplasmic reticulum; Integrated stress response; MRNA; Processing body; Stress granule; Translation

DOI:  https://doi.org/10.1016/j.semcdb.2022.09.013
Nucleic Acids Res. 2022 Oct 03. pii: gkac805. [Epub ahead of print]

Probing protein ubiquitination in live cells.

Weihua Qin, Clemens Steinek, Ksenia Kolobynina, Ignasi Forné, Axel Imhof, M Cristina Cardoso, Heinrich Leonhardt.

The reversible attachment of ubiquitin governs the interaction, activity and degradation of proteins whereby the type and target of this conjugation determine the biological response. The investigation of this complex and multi-faceted protein ubiquitination mostly relies on painstaking biochemical analyses. Here, we employ recombinant binding domains to probe the ubiquitination of proteins in living cells. We immobilize GFP-fused proteins of interest at a distinct cellular structure and detect their ubiquitination state with red fluorescent ubiquitin binders. With this ubiquitin fluorescent three-hybrid (ubiF3H) assay we identified HP1β as a novel ubiquitination target of UHRF1. The use of linkage specific ubiquitin binding domains enabled the discrimination of K48 and K63 linked protein ubiquitination. To enhance signal-to-noise ratio, we implemented fluorescence complementation (ubiF3Hc) with split YFP. Using in addition a cell cycle marker we could show that HP1β is mostly ubiquitinated by UHRF1 during S phase and deubiquitinated by the protease USP7. With this complementation assay we could also directly detect the ubiquitination of the tumor suppressor p53 and monitor its inhibition by the anti-cancer drug Nutlin-3. Altogether, we demonstrate the utility of the ubiF3H assay to probe the ubiquitination of specific proteins and to screen for ligases, proteases and small molecules controlling this posttranslational modification.

DOI: https://doi.org/10.1093/nar/gkac805
Autophagy. 2022 Oct 05.

A Noncanonical Autophagy Function of ATG9A for Golgi Integrity and Dynamics.

Yuanyuan Meng, Qian Luo, Quan Chen, Yushan Zhu.

  In mammalian cells, the Golgi apparatus serves as the central hub for membrane trafficking. Notably, the membrane trafficking and Golgi integrity are tightly regulated by reversible post-translational modifications, such as glycosylation, phosphorylation and ubiquitination. Nonetheless, how the Golgi apparatus responses to stress to ensure appropriate membrane assembly and distribution of cargo is poorly understood. The Golgi resident protein ATG9A is the only multi-spanning membrane protein in the ATG family, and has been demonstrated to traffic through the plasma membrane, endosomes, and Golgi to deliver materials for the initiation of macroautophagy/autophagy. Our recent work reveals a noncanonical function of ATG9A for Golgi dynamics and identifies a pathway for sensing Golgi stress via the MARCHF9-ATG9A axis.

Keywords:  ATG9A; Golgi dynamics; Golgi stress response; MARCHF9; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2022.2131244
Nat Commun. 2022 Oct 05. 13(1): 5874

Thermosensation in Caenorhabditis elegans is linked to ubiquitin-dependent protein turnover via insulin and calcineurin signalling.

Alexandra Segref, Kavya L Vakkayil, Tsimafei Padvitski, Qiaochu Li, Virginia Kroef, Jakob Lormann, Lioba Körner, Fabian Finger, Thorsten Hoppe.

Organismal physiology and survival are influenced by environmental conditions and linked to protein quality control. Proteome integrity is achieved by maintaining an intricate balance between protein folding and degradation. In Caenorhabditis elegans, acute heat stress determines cell non-autonomous regulation of chaperone levels. However, how the perception of environmental changes, including physiological temperature, affects protein degradation remains largely unexplored. Here, we show that loss-of-function of dyf-1 in Caenorhabditis elegans associated with dysfunctional sensory neurons leads to defects in both temperature perception and thermal adaptation of the ubiquitin/proteasome system centered on thermosensory AFD neurons. Impaired perception of moderate temperature changes worsens ubiquitin-dependent proteolysis in intestinal cells. Brain-gut communication regulating protein turnover is mediated by upregulation of the insulin-like peptide INS-5 and inhibition of the calcineurin-regulated forkhead-box transcription factor DAF-16/FOXO. Our data indicate that perception of ambient temperature and its neuronal integration is important for the control of proteome integrity in complex organisms.

DOI: https://doi.org/10.1038/s41467-022-33467-7
Cell. 2022 Sep 30. pii: S0092-8674(22)01123-0. [Epub ahead of print]

Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

Qiaoxia Zheng, Yong Chen, Di Chen, Hongyu Zhao, Yun Feng, Quan Meng, Yan Zhao, Hong Zhang.

  The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca2+ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.

Keywords:  ATG9; Ca(2+) transient; EI24; ER; FIP200; autophagosome formation; liquid-liquid phase separation; lysosome

DOI:  https://doi.org/10.1016/j.cell.2022.09.001
Cell Rep. 2022 Oct 04. pii: S2211-1247(22)01276-1. [Epub ahead of print]41(1): 111435

Destabilization of TP53 by USP10 is essential for neonatal autophagy and survival.

Hongchang Li, Chaonan Li, Wenjing Zhai, Xin Zhang, Lei Li, Bo Wu, Biyue Yu, Pengfei Zhang, Jie Li, Chun-Ping Cui, Lingqiang Zhang.

  Autophagy is essential for the maintenance of energy homeostasis and for survival during the neonatal starvation period. At birth, the trans-placental nutrient supply is suddenly interrupted, and neonates adapt to this adverse circumstance by activating autophagy. However, the mechanisms underlying the precise regulation of neonatal autophagy remain undefined. Here, we show that the destabilization of TP53 by the deubiquitylase ubiquitin-specific peptidase 10 (USP10) is essential for neonatal autophagy and survival. Usp10 deficiency results in decreased E3 ligase activity of MDM2 and accumulation of cytoplasmic TP53, which interferes with the conjugation of ATG12 and ATG5, the key autophagy-related genes, and ultimately inhibits autophagy in neonatal mice. Combined deletion of Tp53 and Usp10 recovers the nutrition supply and rescues the death phenotype of Usp10-deficient neonates. These findings reveal a role of the USP10-MDM2-TP53 axis in nutrient homeostasis and neonatal viability and provide insights into the long-perplexing mechanism by which cytoplasmic TP53 inhibits autophagy.

Keywords:  CP: Developmental biology; CP: Molecular biology; TP53; USP10; autophagy; deubiquitination; neonatal survival

DOI:  https://doi.org/10.1016/j.celrep.2022.111435
Biophys J. 2022 Oct 05. pii: S0006-3495(22)00825-6. [Epub ahead of print]

Model for ring closure in ER tubular network dynamics.

Ben Zucker, Gonen Golani, Michael M Kozlov.

Tubular networks of the endoplasmic reticulum (ER) are dynamic structures whose steady-state conformations are maintained by a balance between the persistent generation and vanishing of the network elements. While factors producing the ER tubules and inter-tubular junctions have been investigated, the mechanisms behind their elimination remained unknown. Here we addressed the ER ring closure, the process resulting in the tubule and junction removal through constriction of the network unit-cells into junctional knots followed by the knot remodeling into regular junctions. We considered the ring closure to be driven by the tension existing in ER membranes. We based our consideration on the notion of Gibbs' thermodynamic tension and reviewed its relationship to other tension definitions used in the literature. We modeled, computationally, the structures of the junctional knots containing internal nanopores and analyzed their tension dependence. We analyzed the process of the pore sealing through membrane fission resulting in formation of regular junctions. Considering the hemi-fission as the rate-limiting stage of the fission reaction, we evaluated the membrane tensions guarantying the spontaneous character of the pore sealing. We concluded that feasible membrane tensions explain all stages of the ER ring closure.

DOI: https://doi.org/10.1016/j.bpj.2022.10.005
Sci Adv. 2022 Oct 07. 8(40): eadd4253

DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on protein substrates.

Kang Zhu, Marcin J Suskiewicz, Andrea Hloušek-Kasun, Hervé Meudal, Andreja Mikoč, Vincent Aucagne, Dragana Ahel, Ivan Ahel.

Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged. Here, we show that DELTEX E3 ligases specifically target the 3' hydroxyl of the adenosine diphosphate (ADP)-ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3~ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2~ubiquitin onto a substrate. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad-like manner. In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells.

DOI: https://doi.org/10.1126/sciadv.add4253
Nat Commun. 2022 Oct 06. 13(1): 5884

Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry.

Tom Dixon, Derek MacPherson, Barmak Mostofian, Taras Dauzhenka, Samuel Lotz, Dwight McGee, Sharon Shechter, Utsab R Shrestha, Rafal Wiewiora, Zachary A McDargh, Fen Pei, Rajat Pal, João V Ribeiro, Tanner Wilkerson, Vipin Sachdeva, Ning Gao, Shourya Jain, Samuel Sparks, Yunxing Li, Alexander Vinitsky, Xin Zhang, Asghar M Razavi, István Kolossváry, Jason Imbriglio, Artem Evdokimov, Louise Bergeron, Wenchang Zhou, Jagat Adhikari, Benjamin Ruprecht, Alex Dickson, Huafeng Xu, Woody Sherman, Jesus A Izaguirre.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

DOI: https://doi.org/10.1038/s41467-022-33575-4
Nat Commun. 2022 Oct 04. 13(1): 5845

Autophagy induction promoted by m6A reader YTHDF3 through translation upregulation of FOXO3 mRNA.

WeiChao Hao, MeiJuan Dian, Ying Zhou, QiuLing Zhong, WenQian Pang, ZiJian Li, YaYan Zhao, JiaCheng Ma, XiaoLin Lin, RenRu Luo, YongLong Li, JunShuang Jia, HongFen Shen, ShiHao Huang, GuanQi Dai, JiaHong Wang, Yan Sun, Dong Xiao.

Autophagy is crucial for maintaining cellular energy homeostasis and for cells to adapt to nutrient deficiency, and nutrient sensors regulating autophagy have been reported previously. However, the role of eiptranscriptomic modifications such as m6A in the regulation of starvation-induced autophagy is unclear. Here, we show that the m6A reader YTHDF3 is essential for autophagy induction. m6A modification is up-regulated to promote autophagosome formation and lysosomal degradation upon nutrient deficiency. METTL3 depletion leads to a loss of functional m6A modification and inhibits YTHDF3-mediated autophagy flux. YTHDF3 promotes autophagy by recognizing m6A modification sites around the stop codon of FOXO3 mRNA. YTHDF3 also recruits eIF3a and eIF4B to facilitate FOXO3 translation, subsequently initiating autophagy. Overall, our study demonstrates that the epitranscriptome regulator YTHDF3 functions as a nutrient responder, providing a glimpse into the post-transcriptional RNA modifications that regulate metabolic homeostasis.

DOI: https://doi.org/10.1038/s41467-022-32963-0
Commun Biol. 2022 Oct 05. 5(1): 1060

Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle.

Nirajan Neupane, Jayasimman Rajendran, Jouni Kvist, Sandra Harjuhaahto, Bowen Hu, Veijo Kinnunen, Yang Yang, Anni I Nieminen, Henna Tyynismaa.

Effective protein import from cytosol is critical for mitochondrial functions and metabolic regulation. We describe here the mammalian muscle-specific and systemic consequences to disrupted mitochondrial matrix protein import by targeted deletion of the mitochondrial HSP70 co-chaperone GRPEL1. Muscle-specific loss of GRPEL1 caused rapid muscle atrophy, accompanied by shut down of oxidative phosphorylation and mitochondrial fatty acid oxidation, and excessive triggering of proteotoxic stress responses. Transcriptome analysis identified new responders to mitochondrial protein import toxicity, such as the neurological disease-linked intermembrane space protein CHCHD10. Besides communication with ER and nucleus, we identified crosstalk of distressed mitochondria with peroxisomes, in particular the induction of peroxisomal Acyl-CoA oxidase 2 (ACOX2), which we propose as an ATF4-regulated peroxisomal marker of integrated stress response. Metabolic profiling indicated fatty acid enrichment in muscle, a shift in TCA cycle intermediates in serum and muscle, and dysregulated bile acids. Our results demonstrate the fundamental importance of GRPEL1 and provide a robust model for detecting mammalian inter-organellar and systemic responses to impaired mitochondrial matrix protein import and folding.

DOI: https://doi.org/10.1038/s42003-022-04034-z
Nucleic Acids Res. 2022 Oct 06. pii: gkac843. [Epub ahead of print]

DDX17 is required for efficient DSB repair at DNA:RNA hybrid deficient loci.

Aldo S Bader, Janna Luessing, Ben R Hawley, George L Skalka, Wei-Ting Lu, Noel F Lowndes, Martin Bushell.

Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that have low levels of pre-existing hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.

DOI: https://doi.org/10.1093/nar/gkac843
Cell Death Discov. 2022 Oct 04. 8(1): 407

Proteotoxic stress-induced apoptosis in cancer cells: understanding the susceptibility and enhancing the potency.

Luca Iuliano, Emiliano Dalla, Raffaella Picco, Showmeya Mallavarapu, Martina Minisini, Eleonora Malavasi, Claudio Brancolini.

Leiomyosarcoma (LMS) is aggressive cancer with few therapeutic options. LMS cells are more sensitive to proteotoxic stress compared to normal smooth muscle cells. We used small compound 2c to induce proteotoxic stress and compare the transcriptomic adaptations of immortalized human uterine smooth muscle cells (HUtSMC) and LMS cells SK-UT-1. We found that the expression of the heat shock proteins (HSPs) gene family is upregulated with higher efficiency in normal cells. In contrast, the upregulation of BH3-only proteins is higher in LMS cells. HSF1, the master regulator of HSP transcription, is sequestered into transcriptionally incompetent nuclear foci only in LMS cells, which explains the lower HSP upregulation. We also found that several compounds can enhance the cell death response to proteotoxic stress. Specifically, when low doses were used, an inhibitor of salt-inducible kinases (SIKs) and the inhibitor of IRE1α, a key element of the unfolded protein response (UPR), support proteotoxic-induced cell death with strength in LMS cells and without effects on the survival of normal cells. Overall, our data provide an explanation for the higher susceptibility of LMS cells to proteotoxic stress and suggest a potential option for co-treatment strategies.

DOI: https://doi.org/10.1038/s41420-022-01202-2
Oncogene. 2022 Oct 06.

Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance.

Changxu Wang, Qilai Long, Qiang Fu, Qixia Xu, Da Fu, Yan Li, Libin Gao, Jianming Guo, Xiaoling Zhang, Eric W-F Lam, Judith Campisi, Yu Sun.

The tumor microenvironment (TME) represents a milieu enabling cancer cells to develop malignant properties, while concerted interactions between cancer and stromal cells frequently shape an "activated/reprogramed" niche to accelerate pathological progression. Here we report that a soluble factor epiregulin (EREG) is produced by senescent stromal cells, which non-cell-autonomously develop the senescence-associated secretory phenotype (SASP) upon DNA damage. Genotoxicity triggers EREG expression by engaging NF-κB and C/EBP, a process supported by elevated chromatin accessibility and increased histone acetylation. Stromal EREG reprograms the expression profile of recipient neoplastic cells in a paracrine manner, causing upregulation of MARCHF4, a membrane-bound E3 ubiquitin ligase involved in malignant progression, specifically drug resistance. A combinational strategy that empowers EREG-specific targeting in treatment-damaged TME significantly promotes cancer therapeutic efficacy in preclinical trials, achieving response indices superior to those of solely targeting cancer cells. In clinical oncology, EREG is expressed in tumor stroma and handily measurable in circulating blood of cancer patients post-chemotherapy. This study establishes EREG as both a targetable SASP factor and a new noninvasive biomarker of treatment-damaged TME, thus disclosing its substantial value in translational medicine.

DOI: https://doi.org/10.1038/s41388-022-02476-7
Nat Biotechnol. 2022 Oct 06.

Post-translational modifications reshape the antigenic landscape of the MHC I immunopeptidome in tumors.

Assaf Kacen, Aaron Javitt, Matthias P Kramer, David Morgenstern, Tomer Tsaban, Merav D Shmueli, Guo Ci Teo, Felipe da Veiga Leprevost, Eilon Barnea, Fengchao Yu, Arie Admon, Lea Eisenbach, Yardena Samuels, Ora Schueler-Furman, Yishai Levin, Alexey I Nesvizhskii, Yifat Merbl.

Post-translational modification (PTM) of antigens provides an additional source of specificities targeted by immune responses to tumors or pathogens, but identifying antigen PTMs and assessing their role in shaping the immunopeptidome is challenging. Here we describe the Protein Modification Integrated Search Engine (PROMISE), an antigen discovery pipeline that enables the analysis of 29 different PTM combinations from multiple clinical cohorts and cell lines. We expanded the antigen landscape, uncovering human leukocyte antigen class I binding motifs defined by specific PTMs with haplotype-specific binding preferences and revealing disease-specific modified targets, including thousands of new cancer-specific antigens that can be shared between patients and across cancer types. Furthermore, we uncovered a subset of modified peptides that are specific to cancer tissue and driven by post-translational changes that occurred in the tumor proteome. Our findings highlight principles of PTM-driven antigenicity, which may have broad implications for T cell-mediated therapies in cancer and beyond.

DOI: https://doi.org/10.1038/s41587-022-01464-2
Cell. 2022 Sep 22. pii: S0092-8674(22)01124-2. [Epub ahead of print]

X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.

Yan Yan, Xinming Wang, Dale Chaput, Min-Kyoo Shin, Yeojung Koh, Li Gan, Andrew A Pieper, Jung-A A Woo, David E Kang.

  Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.

Keywords:  Alzheimer’s; DUB; USP11; X-inactivation; acetylation; long-term potentiation; spatial memory; synaptic plasticity; tauopathy; ubiquitin

DOI:  https://doi.org/10.1016/j.cell.2022.09.002
Sci Adv. 2022 Oct 07. 8(40): eabo3932

Aging compromises human islet beta cell function and identity by decreasing transcription factor activity and inducing ER stress.

Shristi Shrestha, Galina Erikson, James Lyon, Aliya F Spigelman, Austin Bautista, Jocelyn E Manning Fox, Cristiane Dos Santos, Maxim Shokhirev, Jean-Philippe Cartailler, Martin W Hetzer, Patrick E MacDonald, Rafael Arrojo E Drigo.

Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.

DOI: https://doi.org/10.1126/sciadv.abo3932
Elife. 2022 Oct 04. pii: e75978. [Epub ahead of print]11

Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with challenges to proteostasis.

Julie Paxman, Zhen Zhou, Richard O'Laughlin, Yuting Liu, Yang Li, Wanying Tian, Hetian Su, Yanfei Jiang, Shayna E Holness, Elizabeth Stasiowski, Lev S Tsimring, Lorraine Pillus, Jeff Hasty, Nan Hao.

  Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of S. cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.

Keywords:  S. cerevisiae; cell biology; computational biology; systems biology

DOI:  https://doi.org/10.7554/eLife.75978