bims-proteo 2022-03-20 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022–03–20
forty papers selected by
Eric Chevet, INSERM

Endoplasmic reticulum-unfolded protein response pathway modulates the cellular response to mitochondrial proteotoxic stress.
Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18.
The unfolded protein response transducer IRE1α promotes reticulophagy in podocytes.
A trio of ubiquitin ligases sequentially drives ubiquitylation and autophagic degradation of dysfunctional yeast proteasomes.
Generation of Monoubiquitin and K63-Linked Polyubiquitin Chains for Protein Interaction Studies.
Quality control of protein complex assembly by the ubiquitin-proteasome system.
The Function, Structure, and Origins of the ER Membrane Protein Complex.
The needs of a synapse-How local organelles serve synaptic proteostasis.
Linear ubiquitin as a common regulator of cellular stress.
Ribosome-nascent chain interaction regulates N-terminal protein modification.
Self-association status-dependent inactivation of the endoplasmic reticulum stress sensor Ire1 by C-terminal tagging with artificial peptides.
Assembly mechanism of early Hsp90-Cdc37-kinase complexes.
Phosphatidic acid suppresses autophagy through competitive inhibition by binding GAPC (glyceraldehyde-3-phosphate dehydrogenase) and PGK (phosphoglycerate kinase) proteins.
eIF3a regulation of mTOR signaling and translational control via HuR in cellular response to DNA damage.
HSF2 cooperates with HSF1 to drive a transcriptional program critical for the malignant state.
Material properties of phase-separated TFEB condensates regulate the autophagy-lysosome pathway.
Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics.
Role of ER stress inhibitors in the management of diabetes.
Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.
TCA cycle off, ATF4 on for metabolic homeostasis.
Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia.
Profiling of diverse tumor types establishes the broad utility of VHL-based ProTaCs and triages candidate ubiquitin ligases.
Multi-ubiquitination of TRPV4 reduces channel activity independent of surface localization.
Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates.
Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.
Lysosomal Ca2+-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress.
Pseudouridine-modified tRNA fragments repress aberrant protein synthesis and predict leukaemic progression in myelodysplastic syndrome.
SIAH ubiquitin E3 ligases as modulators of inflammatory gene expression.
Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis.
Chemical Biology Tools for Protein Lysine Acylation.
Mechanistic insights into the subversion of the linear ubiquitin chain assembly complex by the E3 ligase IpaH1.4 of Shigella flexneri.
Loss of RUBCN/rubicon in adipocytes mediates the upregulation of autophagy to promote the fasting response.
Role of XBP1 in regulating the progression of non-alcoholic steatohepatitis.
Hsp104 N-terminal Domain Interaction with Substrates Plays a Regulatory Role in Protein Disaggregation.
Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration.
Copper Induces Protein Aggregation, a Toxic Process Compensated by Molecular Chaperones.
Mapping the sequence specificity of heterotypic amyloid interactions enables the identification of aggregation modifiers.
Lysosomal inhibition sensitizes TMEM16A-expressing cancer cells to chemotherapy.
Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding.
M6A RNA Methylation Regulates Histone Ubiquitination to Support Cancer Growth and Progression.

Cell Stress Chaperones. 2022 Mar 16.

Endoplasmic reticulum-unfolded protein response pathway modulates the cellular response to mitochondrial proteotoxic stress.

Rajasri Sarkar, Kannan Boosi Narayana Rao, Mainak Pratim Jha, Koyeli Mapa.

  Mitochondria and endoplasmic reticulum (ER) remain closely tethered by contact sites to maintain unhindered biosynthetic, metabolic, and signalling functions. Apart from its constituent proteins, contact sites localize ER-unfolded protein response (UPR) sensors like Ire1 and PERK, indicating the importance of ER-mitochondria communication during stress. In the mitochondrial sub-compartment-specific proteotoxic model of yeast, Saccharomyces cerevisiae, we show that an intact ER-UPR pathway is important in stress tolerance of mitochondrial intermembrane space (IMS) proteotoxic stress, while disrupting the pathway is beneficial during matrix stress. Deletion of IRE1 and HAC1 leads to accumulation of misfolding-prone proteins in mitochondrial IMS indicating the importance of intact ER-UPR pathway in enduring mitochondrial IMS proteotoxic stresses. Although localized proteotoxic stress within mitochondrial IMS does not induce ER-UPR, its artificial activation helps cells to better withstand the IMS proteotoxicity. Furthermore, overexpression of individual components of ER-mitochondria contact sites is found to be beneficial for general mitochondrial proteotoxic stress, in an Ire1-Hac1-independent manner.

Keywords:  ER stress; ER-mitochondria contact sites; Mito-UPR; Protein homeostasis; Proteotoxic stress; Unfolded protein response

DOI:  https://doi.org/10.1007/s12192-022-01264-2
Proc Natl Acad Sci U S A. 2022 Mar 22. 119(12): e2122657119

Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18.

Ojore Benedict Valentine Oka, Arvin Shedrach Pierre, Marie Anne Pringle, Wanida Tungkum, Zhenbo Cao, Bethany Fleming, Neil John Bulleid.

  SignificanceMembrane and secretory proteins are synthesized in the endoplasmic reticulum (ER). Perturbations to ER function disrupts protein folding, causing misfolded proteins to accumulate, a condition known as ER stress. Cells adapt to stress by activating the unfolded protein response (UPR), which ultimately restores proteostasis. A key player in the UPR response is ATF6α, which requires release from ER retention and modulation of its redox status during activation. Here, we report that ER stress promotes formation of a specific ATF6α dimer, which is preferentially trafficked to the Golgi for processing. We show that ERp18 regulates ATF6α by mitigating its dimerization and trafficking to the Golgi and identify redox-dependent oligomerization of ATF6α as a key mechanism regulating its function during the UPR.

Keywords:  ATF6; ER stress; proteostasis; unfolded protein response

DOI:  https://doi.org/10.1073/pnas.2122657119
Biochim Biophys Acta Mol Basis Dis. 2022 Mar 15. pii: S0925-4439(22)00054-0. [Epub ahead of print] 166391

The unfolded protein response transducer IRE1α promotes reticulophagy in podocytes.

José R Navarro-Betancourt, Joan Papillon, Julie Guillemette, Chen-Fang Chung, Takao Iwawaki, Andrey V Cybulsky.

  Glomerular diseases involving podocyte/glomerular epithelial cell (GEC) injury feature protein misfolding and endoplasmic reticulum (ER) stress. Inositol-requiring enzyme 1α (IRE1α) mediates chaperone production and autophagy during ER stress. We examined the role of IRE1α in selective autophagy of the ER (reticulophagy). Control and IRE1α knockout (KO) GECs were incubated with tunicamycin to induce ER stress and subjected to proteomic analysis. This showed IRE1α-dependent upregulation of secretory pathway mediators, including the coat protein complex II component Sec23B. Tunicamycin enhanced expression of Sec23B and the reticulophagy adaptor reticulon-3-long (RTN3L) in control, but not IRE1α KO GECs. Knockdown of Sec23B reduced autophagosome formation in response to ER stress. Tunicamycin stimulated colocalization of autophagosomes with Sec23B and RTN3L in an IRE1α-dependent manner. Similarly, during ER stress, glomerular α5 collagen IV colocalized with RTN3L and autophagosomes. Degradation of RTN3L and collagen IV increased in response to tunicamycin, and the turnover was blocked by deletion of IRE1α; thus, the IRE1α pathway promotes RTN3L-mediated reticulophagy and collagen IV may be an IRE1α-dependent reticulophagy substrate. In experimental glomerulonephritis, expression of Sec23B, RTN3L, and LC3-II increased in glomeruli of control mice, but not in podocyte-specific IRE1α KO littermates. In conclusion, during ER stress, IRE1α redirects a subset of Sec23B-positive vesicles to deliver RTN3L-coated ER fragments to autophagosomes. Reticulophagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.

Keywords:  Autophagy; Collagen IV; ERphagy; Endoplasmic reticulum stress; Reticulon-3; Sec23B

DOI:  https://doi.org/10.1016/j.bbadis.2022.166391
Cell Rep. 2022 Mar 15. pii: S2211-1247(22)00276-5. [Epub ahead of print]38(11): 110535

A trio of ubiquitin ligases sequentially drives ubiquitylation and autophagic degradation of dysfunctional yeast proteasomes.

Richard S Marshall, Richard D Vierstra.

  As central effectors of ubiquitin (Ub)-mediated proteolysis, proteasomes are regulated at multiple levels, including degradation of unwanted or dysfunctional particles via autophagy (termed proteaphagy). In yeast, inactive proteasomes are exported from the nucleus, sequestered into cytoplasmic aggresomes via the Hsp42 chaperone, extensively ubiquitylated, and then tethered to the expanding phagophore by the autophagy receptor Cue5. Here, we demonstrate the need for ubiquitylation driven by the trio of Ub ligases (E3s), San1, Rsp5, and Hul5, which together with their corresponding E2s work sequentially to promote nuclear export and Cue5 recognition. Whereas San1 functions prior to nuclear export, Rsp5 and Hul5 likely decorate aggresome-localized proteasomes in concert. Ultimately, topologically complex Ub chain(s) containing both K48 and K63 Ub-Ub linkages are assembled, mainly on the regulatory particle, to generate autophagy-competent substrates. Because San1, Rsp5, Hul5, Hsp42, and Cue5 also participate in general proteostasis, proteaphagy likely engages a fundamental mechanism for eliminating inactive/misfolded proteins.

Keywords:  CP: Molecular Biology; aggresome; autophagy; core protease; proteaphagy; proteasome; regulatory particle; ubiquitin; ubiquitin ligase; yeast

DOI:  https://doi.org/10.1016/j.celrep.2022.110535
Methods Mol Biol. 2022 ;2444 271-282

Generation of Monoubiquitin and K63-Linked Polyubiquitin Chains for Protein Interaction Studies.

Rita Anoh, Kate A Burke, Dhane P Schmelyun, Patrick M Lombardi.

  Ubiquitylation is a posttranslational modification that utilizes protein-protein binding interactions to regulate cellular processes. In ubiquitin signaling, a vast array of mono- and polyubiquitin modifications to substrate proteins are recognized by a diverse group of ubiquitin-binding proteins. Identifying ubiquitin-binding proteins and characterizing their binding properties is necessary for understanding the structural basis of ubiquitin signaling. This chapter provides a means of studying ubiquitin-binding interactions in vitro by describing how to generate monoubiquitin and K63-linked polyubiquitin chains and perform pull-down assays with ubiquitin-binding proteins, which is of particular relevance for DNA damage and other signaling pathways.

Keywords:  Cellular signaling; Polyubiquitin chains; Pull-down assay; Ubiquitin; Ubiquitin-activating enzyme; Ubiquitin-binding protein; Ubiquitin-conjugating enzyme; Ubiquitin-conjugation reaction

DOI:  https://doi.org/10.1007/978-1-0716-2063-2_16
Trends Cell Biol. 2022 Mar 14. pii: S0962-8924(22)00038-1. [Epub ahead of print]

Quality control of protein complex assembly by the ubiquitin-proteasome system.

Carlos Pla-Prats, Nicolas H Thomä.

  The majority of human proteins operate as multimeric complexes with defined compositions and distinct architectures. How the assembly of these complexes is surveyed and how defective complexes are recognized is just beginning to emerge. In eukaryotes, over 600 E3 ubiquitin ligases form part of the ubiquitin-proteasome system (UPS) which detects structural characteristics in its target proteins and selectively induces their degradation. The UPS has recently been shown to oversee key quality control steps during the assembly of protein complexes. We review recent findings on how E3 ubiquitin ligases regulate protein complex assembly and highlight unanswered questions relating to their mechanism of action.

Keywords:  AQC; UPS; assembly; protein complex; quality control; ubiquitin

DOI:  https://doi.org/10.1016/j.tcb.2022.02.005
Annu Rev Biochem. 2022 Feb 14.

The Function, Structure, and Origins of the ER Membrane Protein Complex.

Ramanujan S Hegde.

The endoplasmic reticulum (ER) is the site of membrane protein insertion, folding, and assembly in eukaryotes. Over the past few years, a combination of genetic and biochemical studies have implicated an abundant factor termed the ER membrane protein complex (EMC) in several aspects of membrane protein biogenesis. This large nine-protein complex is built around a deeply conserved core formed by the EMC3-EMC6 subcomplex. EMC3 belongs to the universally conserved Oxa1 superfamily of membrane protein transporters, whereas EMC6 is an ancient, widely conserved obligate partner. EMC has an established role in the insertion of transmembrane domains (TMDs) and less understood roles during the later steps of membrane protein folding and assembly. Several recent structures suggest hypotheses about the mechanism(s) of TMD insertion by EMC, with various biochemical and proteomics studies beginning to reveal the range of EMC's membrane protein substrates. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-biochem-032620-104553
EMBO J. 2022 Mar 14. e110057

The needs of a synapse-How local organelles serve synaptic proteostasis.

Katarzyna M Grochowska, Maria Andres-Alonso, Anna Karpova, Michael R Kreutz.

  Synaptic function crucially relies on the constant supply and removal of neuronal membranes. The morphological complexity of neurons poses a significant challenge for neuronal protein transport since the machineries for protein synthesis and degradation are mainly localized in the cell soma. In response to this unique challenge, local micro-secretory systems have evolved that are adapted to the requirements of neuronal membrane protein proteostasis. However, our knowledge of how neuronal proteins are synthesized, trafficked to membranes, and eventually replaced and degraded remains scarce. Here, we review recent insights into membrane trafficking at synaptic sites and into the contribution of local organelles and micro-secretory pathways to synaptic function. We describe the role of endoplasmic reticulum specializations in neurons, Golgi-related organelles, and protein complexes like retromer in the synthesis and trafficking of synaptic transmembrane proteins. We discuss the contribution of autophagy and of proteasome-mediated and endo-lysosomal degradation to presynaptic proteostasis and synaptic function, as well as nondegradative roles of autophagosomes and lysosomes in signaling and synapse remodeling. We conclude that the complexity of neuronal cyto-architecture necessitates long-distance protein transport that combines degradation with signaling functions.

Keywords:  Golgi satellites; autophagy; lysosomes; secretory trafficking

DOI:  https://doi.org/10.15252/embj.2021110057
FEBS J. 2022 Mar 16.

Linear ubiquitin as a common regulator of cellular stress.

Nieves Peltzer.

  Linear or M1-ubiquitination (Ub) is required for optimal NF-kB activation and for cell death inhibition. Using Drosophila as a model organism, Aalto et al. found that hypoxia, oxidative and mechanical stress induced M1-Ub by the HOIP homolog, LUBEL. Increased M1-Ub had a protective function driven by activation of the NF-κB transcription factor Relish via the Immune deficiency pathway (Imd). This protective M1-Ub was also induced upon cellular stress in colorectal cancer cells. Collectively, they propose that M1-Ub is a conserved, common response to different forms of stresses. These findings may have important implications for the use of HOIP inhibitors for cancer treatment. Comment on: https://doi.org/10.1111/febs.16425.

Keywords:  LUBAC; LUBEL; NF-κB; cell death; hypoxia; oxidative stress; stress response

DOI:  https://doi.org/10.1111/febs.16427
J Mol Biol. 2022 Mar 09. pii: S0022-2836(22)00109-7. [Epub ahead of print] 167535

Ribosome-nascent chain interaction regulates N-terminal protein modification.

Chien-I Yang, Jiwoo Kim, Shu-Ou Shan.

  Numerous proteins initiate their folding, localization, and modifications early during translation, and emerging data show that the ribosome actively participates in diverse protein biogenesis pathways. Here we show that the ribosome imposes an additional layer of substrate selection during N-terminal methionine excision (NME), an essential protein modification in bacteria. Biochemical analyses show that cotranslational NME is exquisitely sensitive to a hydrophobic signal sequence or transmembrane domain near the N-terminus of the nascent polypeptide. The ability of the nascent chain to access the active site of NME enzymes dictates NME efficiency, which is inhibited by confinement of the nascent chain on the ribosome surface and exacerbated by signal recognition particle. In vivo measurements corroborate the inhibition of NME by an N-terminal hydrophobic sequence, suggesting the retention of formylmethionine on a substantial fraction of the secretory and membrane proteome. Our work demonstrates how molecular features of a protein regulate its cotranslational modification and highlights the active participation of the ribosome in protein biogenesis pathways via interactions of the ribosome surface with the nascent protein.

Keywords:  N-terminal protein modification; cotranslational protein biogenesis; methionine aminopeptidase; peptide deformylase; ribosome

DOI:  https://doi.org/10.1016/j.jmb.2022.167535
Biosci Biotechnol Biochem. 2022 Mar 14. pii: zbac038. [Epub ahead of print]

Self-association status-dependent inactivation of the endoplasmic reticulum stress sensor Ire1 by C-terminal tagging with artificial peptides.

Yuki Ishiwata-Kimata, Tatsuya Hata, Yukio Kimata.

  Upon endoplasmic reticulum (ER) stress, eukaryotic cells commonly induce unfolded protein response (UPR), which is triggered, at least partly, by the ER stress sensor Ire1. Upon ER stress, Ire1 is dimerized or forms oligomeric clusters, resulting in the activation of Ire1 as an endoribonuclease. In ER-stressed Saccharomyces cerevisiae cells, HAC1 mRNA is spliced by Ire1, and then translated into a transcription factor that promotes the UPR. Herein we report that Ire1 tagged artificially with irrelevant peptides at the C terminus is almost completely inactive when only dimerized, while it induced the UPR as well as untagged Ire1 when clustered. This finding suggests a fundamental difference between the dimeric and clustered forms of Ire1. By comparing UPR levels in S. cerevisiae cells carrying artificially peptide-tagged Ire1 to that in cells carrying untagged Ire1, we estimated the self-association status of Ire1 under various ER stress conditions.

Keywords:   Saccharomyces cerevisiae ; endoplasmic reticulum; stress response; unfolded protein response; yeast

DOI:  https://doi.org/10.1093/bbb/zbac038
Sci Adv. 2022 Mar 18. 8(11): eabm9294

Assembly mechanism of early Hsp90-Cdc37-kinase complexes.

Dimitra Keramisanou, M V Vasantha Kumar, Nicole Boose, Rinat R Abzalimov, Ioannis Gelis.

Molecular chaperones have an essential role for the maintenance of a balanced protein homeostasis. Here, we investigate how protein kinases are recruited and loaded to the Hsp90-Cdc37 complex, the first step during Hsp90-mediated chaperoning that leads to enhanced client kinase stability and activation. We show that conformational dynamics of all partners is a critical feature of the underlying loading mechanism. The kinome co-chaperone Cdc37 exists primarily in a dynamic extended conformation but samples a low-populated, well-defined compact structure. Exchange between these two states is maintained in an assembled Hsp90-Cdc37 complex and is necessary for substrate loading. Breathing motions at the N-lobe of a free kinase domain partially expose the kinase segment trapped in the Hsp90 dimer downstream in the cycle. Thus, client dynamics poise for chaperone dependence. Hsp90 is not directly involved during loading, and Cdc37 is assigned the task of sensing clients by stabilizing the preexisting partially unfolded client state.

DOI: https://doi.org/10.1126/sciadv.abm9294
Autophagy. 2022 Mar 15. 1-15

Phosphatidic acid suppresses autophagy through competitive inhibition by binding GAPC (glyceraldehyde-3-phosphate dehydrogenase) and PGK (phosphoglycerate kinase) proteins.

Bin Guan, Yu-Tong Jiang, De-Li Lin, Wen-Hui Lin, Hong-Wei Xue.

  Macroautophagy/autophagy is a finely-regulated process in which cytoplasm encapsulated within transient organelles termed autophagosomes is delivered to lysosomes or vacuoles for degradation. Phospholipids, particularly phosphatidic acid (PA) that functions as a second messenger, play crucial and differential roles in autophagosome formation; however, the underlying mechanism remains largely unknown. Here we demonstrated that PA inhibits autophagy through competitive inhibition of the formation of ATG3 (autophagy-related)-ATG8e and ATG6-VPS34 (vacuolar protein sorting 34) complexes. PA bound to GAPC (glyceraldehyde-3-phosphate dehydrogenase) or PGK (phosphoglycerate kinase) and promoted their interaction with ATG3 or ATG6, which further attenuated the interactions of ATG3-ATG8e or ATG6-VPS34, respectively. Structural and mutational analyses revealed the mechanism of PA binding with GAPCs and PGK3, and that GAPCs or ATG8e competitively interacted with ATG3, and PGK3 or VPS34 competitively interacted with ATG6, at the same binding interface. These results elucidate the molecular mechanism of how PA inhibits autophagy through binding GAPC or PGK3 proteins and expand the understanding of the functional mode of PA, demonstrating the importance of phospholipids in plant autophagy and providing a new perspective for autophagy regulation by phospholipids.Abbreviation: ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; Con A: concanamycin A; ER: endoplasmic reticulum; EZ: elongation zone; FRET-FLIM: fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S-transferase; MDC: monodansylcadaverine; MZ: meristem zone; PA: phosphatidic acid; PAS: phagophore assembly site; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PGK3: phosphoglycerate kinase; PtdIns3K: phosphatidylinositol 3-kinase; PLD: phospholipase D; TEM: transmission electron microscopy; TOR: target of rapamycin; VPS34: vacuolar protein sorting 34; WT: wild type; Y2H: yeast two-hybrid.

Keywords:  Autophagy; autophagy-related protein; competitive inhibition; glyceraldehyde-3-phosphate dehydrogenase; phosphatidic acid; phosphoglycerate kinase

DOI:  https://doi.org/10.1080/15548627.2022.2046449
Oncogene. 2022 Mar 12.

eIF3a regulation of mTOR signaling and translational control via HuR in cellular response to DNA damage.

Shijie Ma, Zizheng Dong, Yanfei Huang, Jing-Yuan Liu, Jian-Ting Zhang.

eIF3a (eukaryotic translation initiation factor 3a), a subunit of the eIF3 complex, has been suggested to play a regulatory role in protein synthesis and in cellular response to DNA-damaging treatments. S6K1 is an effector and a mediator of mTOR complex 1 (mTORC1) in regulating protein synthesis and integrating diverse signals into control of cell growth and response to stress. Here, we show that eIF3a regulates S6K1 activity by inhibiting mTORC1 kinase via regulating Raptor synthesis. The regulation of Raptor synthesis is via eIF3a interaction with HuR (human antigen R) and binding of the eIF3a-HuR complex to the 5'-UTR of Raptor mRNA. Furthermore, mTORC1 may mediate eIF3a function in cellular response to cisplatin by regulating synthesis of NER proteins and NER activity. Taken together, we conclude that the mTOR signaling pathway may also be regulated by translational control and mediate eIF3a regulation of cancer cell response to cisplatin by regulating NER protein synthesis.

DOI: https://doi.org/10.1038/s41388-022-02262-5
Sci Adv. 2022 Mar 18. 8(11): eabj6526

HSF2 cooperates with HSF1 to drive a transcriptional program critical for the malignant state.

Roger S Smith, Seesha R Takagishi, David R Amici, Kyle Metz, Sitaram Gayatri, Milad J Alasady, Yaqi Wu, Sonia Brockway, Stephanie L Taiberg, Natalia Khalatyan, Mikko Taipale, Sandro Santagata, Luke Whitesell, Susan Lindquist, Jeffrey N Savas, Marc L Mendillo.

Heat shock factor 1 (HSF1) is well known for its role in the heat shock response (HSR), where it drives a transcriptional program comprising heat shock protein (HSP) genes, and in tumorigenesis, where it drives a program comprising HSPs and many noncanonical target genes that support malignancy. Here, we find that HSF2, an HSF1 paralog with no substantial role in the HSR, physically and functionally interacts with HSF1 across diverse types of cancer. HSF1 and HSF2 have notably similar chromatin occupancy and regulate a common set of genes that include both HSPs and noncanonical transcriptional targets with roles critical in supporting malignancy. Loss of either HSF1 or HSF2 results in a dysregulated response to nutrient stresses in vitro and reduced tumor progression in cancer cell line xenografts. Together, these findings establish HSF2 as a critical cofactor of HSF1 in driving a cancer cell transcriptional program to support the anabolic malignant state.

DOI: https://doi.org/10.1126/sciadv.abj6526
J Cell Biol. 2022 May 02. pii: e202112024. [Epub ahead of print]221(5):

Material properties of phase-separated TFEB condensates regulate the autophagy-lysosome pathway.

Zheng Wang, Di Chen, Dongshi Guan, Xiaobo Liang, Jianfeng Xue, Hongyu Zhao, Guangtao Song, Jizhong Lou, Yan He, Hong Zhang.

Very little is known about how the material properties of protein condensates assembled via liquid-liquid phase separation (LLPS) are maintained and affect physiological functions. Here we show that liquid-like condensates of the transcription factor TFEB exhibit low fusion propensity in vitro and in living cells. We directly measured the attraction force between droplets, and we characterized the interfacial tension, viscosity, and elasticity of TFEB condensates. TFEB condensates contain rigid interfacial boundaries that govern their interaction behaviors. Several small molecules, including Ro-3306, modify the material properties of TFEB condensates, increasing their size and fusion propensity. These compounds promote lysosomal biogenesis and function in a TFEB-dependent manner without changing its cytoplasmic-nuclear translocation. Ro-3306 promotes autophagy activity, facilitating degradation of toxic protein aggregates. Our study helps explain how protein condensates are maintained as physically separate entities and reveals that the material properties of TFEB condensates can be harnessed to modulate TFEB activity.

DOI: https://doi.org/10.1083/jcb.202112024
Nat Cell Biol. 2022 Mar;24(3): 307-315

Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics.

Maria C Passarelli, Alexandra M Pinzaru, Hosseinali Asgharian, Maria V Liberti, Søren Heissel, Henrik Molina, Hani Goodarzi, Sohail F Tavazoie.

Tumourigenesis and cancer progression require enhanced global protein translation1-3. Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and activity of translational machinery4,5. Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes, including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour-suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase-along with its downstream cognate tRNAs-elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

DOI: https://doi.org/10.1038/s41556-022-00856-5
Eur J Pharmacol. 2022 Mar 12. pii: S0014-2999(22)00154-6. [Epub ahead of print] 174893

Role of ER stress inhibitors in the management of diabetes.

Krishna Prasad M, Sundhar Mohandas, Kunka Mohanram Ramkumar.

  Endoplasmic Reticulum (ER) stress has been established to play a key pathophysiological role in developing metabolic diseases such as Diabetes Mellitus (DM). The complications of DM have been closely associated with deregulation of the unfolded protein response (UPR) signaling pathways, which are critically responsible for restoring homeostasis following ER stress. Chronic ER stress in the background of persistent hyperglycemia, as observed in DM, overwhelms the UPR signaling and commits the cells to apoptosis. The factors such as hyperglycemia, increased reactive oxygen species (ROS), disrupted calcium homeostasis, and overt inflammation serves as major UPR signal transduction pathways, including PKR like ER kinase (PERK), Activating transcription factor 6α/β (ATF6), and Inositol requiring enzyme1α/β (IRE1). The constantly developing understanding of these ER stress mediators has also unraveled their potential as therapeutic targets of small molecules termed ER stress inhibitors. A wide range of both naturally occurring and synthetic compounds have been screened and studied for their properties to inhibit ER stress in various experimental models. This review article elucidates the critical signaling pathways associated with response to ER stress. We shed light on the crosstalk between ER stress mediators with oxidative and inflammatory stress mediators in the background of DM. We extensively summarize the pieces of evidence sourced from preclinical and clinical research about the role of ER stress inhibitors and their pharmacological mechanism of action in alleviating ER stress in diabetes.

Keywords:  Chaperones; Diabetes mellitus; ER stress Inhibitors; Endoplasmic reticulum stress; Therapeutic target; UPR

DOI:  https://doi.org/10.1016/j.ejphar.2022.174893
Autophagy. 2022 Mar 15.

Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

Fangyang Wang, Ying Yang, Gabriel Boudagh, Eeva-Liisa Eskelinen, Daniel J Klionsky, Sami N Malek.

  The discovery of recurrent mutations in subunits and regulators of the vacuolar-type H+-translocating ATPase (V-ATPase) in follicular lymphoma (FL) highlights a role for macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we report on novel mutations in the ER-resident chaperone VMA21, which is involved in V-ATPase assembly in 12% of FL. Mutations in a novel VMA21 hotspot (p.93X) result in the removal of a C-terminal non-canonical ER retrieval signal thus causing VMA21 mislocalization to lysosomes. The resulting impairment in V-ATPase function prevents full lysosomal acidification and function, including impaired pH-dependent protein degradation as shown via lysosomal metabolomics and ultimately causes a degree of amino acid depletion in the cytoplasm. These deficiencies result in compensatory autophagy activation, as measured using multiple complementary assays in human and yeast cells. Of translational significance, the compensatory activation of autophagy creates a dependency for survival for VMA21-mutated primary human FL as shown using inhibitors to ULK1, the proximal autophagy-regulating kinase. Using high-throughput microscopy-based screening assays for autophagy-inhibiting compounds, we identify multiple clinical grade cyclin-dependent kinase inhibitors as promising drugs and thus provide new rationale for innovative clinical trials in FL harboring aberrant V-ATPase.

Keywords:  Follicular lymphoma; VMA21 mutations; autophagy; lysosomal dysfunction; survival

DOI:  https://doi.org/10.1080/15548627.2022.2050663
Trends Biochem Sci. 2022 Mar 12. pii: S0968-0004(22)00065-2. [Epub ahead of print]

TCA cycle off, ATF4 on for metabolic homeostasis.

Durga Mahor, Rishikesh Pandey, Vinay Bulusu.

  Tricarboxylic acid (TCA) cycle is a major hub for catabolic and anabolic reactions, yet cellular metabolic adaptations following its inhibition are largely unknown. Using multi-tiered omics approaches, Ryan et al. have shown convergent activation of the integrated stress response (ISR) through ATF4-mediated rewiring of cellular amino acid and redox metabolic pathways.

Keywords:  ATF4; TCA cycle; fumarate hydratase; glutathione synthesis; integrated stress response; succinate dehydrogenase

DOI:  https://doi.org/10.1016/j.tibs.2022.03.006
Biochim Biophys Acta Mol Basis Dis. 2022 Mar 14. pii: S0925-4439(22)00052-7. [Epub ahead of print] 166389

Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia.

Felipe S A Pimentel, Caio M Machado, Evandro A De-Souza, Caroline Mota Fernandes, Ana Luiza F V De-Queiroz, Guilherme F S Silva, Maurizio Del Poeta, Monica Montero-Lomeli, Claudio A Masuda.

  Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin - an inhibitor of the de novo sphingolipid synthesis pathway - inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.

Keywords:  Galactosemia; Inositol; S. cerevisiae; Sphingolipids; Unfolded protein response

DOI:  https://doi.org/10.1016/j.bbadis.2022.166389
iScience. 2022 Mar 18. 25(3): 103985

Profiling of diverse tumor types establishes the broad utility of VHL-based ProTaCs and triages candidate ubiquitin ligases.

Xin Luo, Ivonne Archibeque, Ken Dellamaggiore, Kate Smither, Oliver Homann, James Russell Lipford, Dane Mohl.

  The success of small molecule therapeutics that promotes degradation of critical cancer targets has fueled an intense effort to mimic this activity with bispecific molecules called PROTACs (proteolysis targeting chimeras). The simultaneous binding of PROTACs to a ligase and target can induce proximity-driven ubiquitination and degradation. VHL and CRBN are the two best characterized PROTAC ligases, but the rules governing their cellular activities remain unclear. To establish these requirements and extend them to new ligases, we screened a panel of 56 cell lines with two potent PROTACs that utilized VHL, MZ1, or CRBN, dBET1 to induce degradation of BRD4. With notable exceptions, MZ1 was broadly active in the panel whereas dBET1 was frequently inactive. A search for predictive biomarkers of PROTAC activity found that expression and mutation of VHL and CRBN were themselves predictors of PROTAC activity in the cell line panel.

Keywords:  Bioengineering; Cancer; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2022.103985
J Biol Chem. 2022 Mar 14. pii: S0021-9258(22)00266-6. [Epub ahead of print] 101826

Multi-ubiquitination of TRPV4 reduces channel activity independent of surface localization.

William H Aisenberg, Brett A McCray, Jeremy M Sullivan, Erika Diehl, Lauren R DeVine, Jonathan Alevy, Anna M Bagnell, Patrice Carr, Jack K Donohue, Benedikt Goretzki, Robert N Cole, Ute A Hellmich, Charlotte J Sumner.

  Ubiquitin-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can also modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multi-ubiquitinated specifically within its cytosolic N- and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 ubiquitin ligase reduced TRPV4 channel activity, but did not decrease plasma membrane abundance. These results demonstrate ubiquitin-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, while increasing mutant TRPV4 ubiquitination partially suppressed channel over-activity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular, flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.

Keywords:  Charcot-Marie-Tooth disease; NEDD4; TRPV4; channel activation; ion channel; plasma membrane; ubiquitination

DOI:  https://doi.org/10.1016/j.jbc.2022.101826
EMBO J. 2022 Mar 17. e108882

Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates.

Janine Hochmair, Christian Exner, Maximilian Franck, Alvaro Dominguez-Baquero, Lisa Diez, Hévila Brognaro, Matthew L Kraushar, Thorsten Mielke, Helena Radbruch, Senthil Kaniyappan, Sven Falke, Eckhard Mandelkow, Christian Betzel, Susanne Wegmann.

  Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

Keywords:  FLIM; MAPT; aggregation; liquid-liquid phase separation; nuclear envelope

DOI:  https://doi.org/10.15252/embj.2021108882
Autophagy. 2022 Mar 16. 1-15

Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.

Devadoss J Samuvel, Li Li, Yasodha Krishnasamy, Monika Gooz, Kenji Takemoto, Patrick M Woster, John J Lemasters, Zhi Zhong.

  Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2-6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo-Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing.Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel.

Keywords:  Acetaldehyde; Alda-1; alcoholic liver disease; mitochondrial depolarization; mitophagy; tacrolimus

DOI:  https://doi.org/10.1080/15548627.2022.2046457
Nat Commun. 2022 Mar 14. 13(1): 1300

Lysosomal Ca2+-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress.

Kihyoun Park, Hyejin Lim, Jinyoung Kim, Yeseong Hwang, Yu Seol Lee, Soo Han Bae, Hyeongseok Kim, Hail Kim, Shin-Wook Kang, Joo Young Kim, Myung-Shik Lee.

Although autophagy is critical for pancreatic β-cell function, the role and mechanism of mitophagy in β-cells are unclear. We studied the role of lysosomal Ca2+ in TFEB activation by mitochondrial or metabolic stress and that of TFEB-mediated mitophagy in β-cell function. Mitochondrial or metabolic stress induced mitophagy through lysosomal Ca2+ release, increased cytosolic Ca2+ and TFEB activation. Lysosomal Ca2+ replenishment by ER- > lysosome Ca2+ refilling was essential for mitophagy. β-cell-specific Tfeb knockout (TfebΔβ-cell) abrogated high-fat diet (HFD)-induced mitophagy, accompanied by increased ROS and reduced mitochondrial cytochrome c oxidase activity or O2 consumption. TfebΔβ-cell mice showed aggravation of HFD-induced glucose intolerance and impaired insulin release. Metabolic or mitochondrial stress induced TFEB-dependent expression of mitophagy receptors including Ndp52 and Optn, contributing to the increased mitophagy. These results suggest crucial roles of lysosomal Ca2+ release coupled with ER- > lysosome Ca2+ refilling and TFEB activation in mitophagy and maintenance of pancreatic β-cell function during metabolic stress.

DOI: https://doi.org/10.1038/s41467-022-28874-9
Nat Cell Biol. 2022 Mar;24(3): 299-306

Pseudouridine-modified tRNA fragments repress aberrant protein synthesis and predict leukaemic progression in myelodysplastic syndrome.

Nicola Guzzi, Sowndarya Muthukumar, Maciej Cieśla, Gabriele Todisco, Phuong Cao Thi Ngoc, Magdalena Madej, Roberto Munita, Serena Fazio, Simon Ekström, Teresa Mortera-Blanco, Monika Jansson, Yasuhito Nannya, Mario Cazzola, Seishi Ogawa, Luca Malcovati, Eva Hellström-Lindberg, Marios Dimitriou, Cristian Bellodi.

Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5' terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5' untranslated region (UTR), including 5' terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5' PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML).

DOI: https://doi.org/10.1038/s41556-022-00852-9
Heliyon. 2022 Mar;8(3): e09029

SIAH ubiquitin E3 ligases as modulators of inflammatory gene expression.

M Lienhard Schmitz, Jan Dreute, Maximilian Pfisterer, Stefan Günther, Michael Kracht, Shashipavan Chillappagari.

  The functionally redundant ubiquitin E3 ligases SIAH1 and SIAH2 have been implicated in the regulation of metabolism and the hypoxic response, while their role in other signal-mediated processes such inflammatory gene expression remains to be defined. Here we have downregulated the expression of both SIAH proteins with specific siRNAs and investigated the functional consequences for IL-1α-induced gene expression. The knockdown of SIAH1/2 modulated the expression of approximately one third of IL-1α-regulated genes. These effects were not due to changes in the NF-κB and MAPK signaling pathways and rather employed further processes including those mediated by the coactivator p300. Most of the proteins encoded by SIAH1/2-regulated genes form a regulatory network of proinflammatory factors. Thus SIAH1/2 proteins function as variable rheostats that control the amplitude rather than the principal activation of the inflammatory gene response.

Keywords:  Inflammatory gene expression; NF-κB; SIAH1; SIAH2; Ubiquitin E3 ligase

DOI:  https://doi.org/10.1016/j.heliyon.2022.e09029
Cell Death Differ. 2022 Mar 16.

Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis.

Xue Han, Chune Ren, Chao Lu, Pengyun Qiao, Tingting Yang, Zhenhai Yu.

MYC as a transcriptional factor plays a crucial role in breast cancer progression. However, the mechanisms underlying MYC deubiquitination in breast cancer are not well defined. Here, we report that OTUB1 is responsible for MYC deubiquitination. OTUB1 could directly deubiquitinate MYC at K323 site, which blocks MYC protein degradation. Moreover, OTUB1 mediated MYC protein stability is also confirmed in OTUB1-knockout mice. Stabilized MYC by OTUB1 promotes its transcriptional activity and induces HK2 expression, which leads to enhance aerobic glycolysis. Therefore, OTUB1 promotes breast tumorigenesis in vivo and in vitro via blocking MYC protein degradation. Taken together, our data identify OTUB1 as a new deubiquitination enzyme for MYC protein degradation, which provides a potential target for breast cancer treatment.

DOI: https://doi.org/10.1038/s41418-022-00971-8
Angew Chem Int Ed Engl. 2022 Mar 18.

Chemical Biology Tools for Protein Lysine Acylation.

Yusheng Xie, Shubo Du, Zhiyang Liu, Min Liu, Zhiqiang Xu, Xiaojie Wang, Jia Xuan Kee, Fan Yi, Hongyan Sun, Shao Q Yao.

  Lysine acylation plays pivotal roles in cell physiology, including DNA transcription and repair, signal transduction, immune defense, metabolism, and many other key cellular processes. Molecular mechanisms of dysregulated lysine acylation are closely involved in the pathophysiological progress of many human diseases, most notably cancers. In recent years, chemical biology tools have become instrumental in studying the function of post-translational modifications (PTMs), identifying new 'writers', 'erasers' and 'readers', and in targeted therapies. Here, we describe key developments in chemical biology approaches that have advanced the study of lysine acylation and its regulatory proteins (2016-2021). We further discuss the discovery of ligands (inhibitors and PROTACs) that are capable of targeting regulators of lysine acylation. Next, we discuss some current challenges of these chemical biology probes and suggest how chemists and biologists can utilize chemical probes with more discriminating capacity. Finally, we suggest some critical considerations in future studies of PTMs from our perspectives.

Keywords:  Chemical probes * Epigenetics * Lysine acylation *Protein machinery * PROTAC

DOI:  https://doi.org/10.1002/anie.202200303
Proc Natl Acad Sci U S A. 2022 Mar 22. 119(12): e2116776119

Mechanistic insights into the subversion of the linear ubiquitin chain assembly complex by the E3 ligase IpaH1.4 of Shigella flexneri.

Jianping Liu, Yaru Wang, Danni Wang, Yingli Wang, Xiaolong Xu, Yuchao Zhang, Ying Li, Mingfang Zhang, Xinyu Gong, Yubin Tang, Liqiang Shen, Miao Li, Lifeng Pan.

  SignificanceShigella flexneri, a deleterious bacterium, causes massive human infection cases and deaths worldwide. To facilitate survival and replication in infected host cells, S. flexneri can secrete two highly similar E3 ligase effectors, IpaH1.4 and IpaH2.5, to subvert the linear ubiquitin chain assembly complex (LUBAC), a key player involved in numerous antibacterial signaling pathways of host cells but with poorly understood mechanisms. In this study, through systematic biochemical and structural characterization, we elucidate the multiple tactics adopted by IpaH1.4/2.5 to disarm the human LUBAC and provide mechanistic insights into the subversion of host LUBAC by IpaH1.4/2.5 of S. flexneri.

Keywords:  HOIL-1L; HOIP; IpaH1.4; LUBAC; ubiquitination

DOI:  https://doi.org/10.1073/pnas.2116776119
Autophagy. 2022 Mar 14. 1-11

Loss of RUBCN/rubicon in adipocytes mediates the upregulation of autophagy to promote the fasting response.

Tadashi Yamamuro, Shuhei Nakamura, Kyosuke Yanagawa, Ayaka Tokumura, Tsuyoshi Kawabata, Atsunori Fukuhara, Hirofumi Teranishi, Maho Hamasaki, Iichiro Shimomura, Tamotsu Yoshimori.

  Upon fasting, adipocytes release their lipids that accumulate in the liver, thus promoting hepatic steatosis and ketone body production. However, the mechanisms underlying this process are not fully understood. In this study, we found that fasting caused a substantial decrease in the adipose levels of RUBCN/rubicon, a negative regulator of macroautophagy/autophagy, along with an increase in autophagy. Adipose-specific rubcn-knockout mice exhibited systemic fat loss that was not accelerated by fasting. Genetic inhibition of autophagy in adipocytes in fasted mice led to a reduction in fat loss, hepatic steatosis, and ketonemia. In terms of mechanism, autophagy decreased the levels of its substrates NCOA1/SRC-1 and NCOA2/TIF2, which are also coactivators of PPARG/PPARγ, leading to a fasting-induced reduction in the mRNA levels of adipogenic genes in adipocytes. Furthermore, RUBCN in adipocytes was degraded through the autophagy pathway, suggesting that autophagic degradation of RUBCN serves as a feedforward system for autophagy induction during fasting. Collectively, we propose that loss of adipose RUBCN promotes a metabolic response to fasting via increasing autophagic activity.

Keywords:  Adipocytes; NCOA1; NCOA2; RUBCN; autophagy; fasting

DOI:  https://doi.org/10.1080/15548627.2022.2047341
J Hepatol. 2022 Mar 12. pii: S0168-8278(22)00140-4. [Epub ahead of print]

Role of XBP1 in regulating the progression of non-alcoholic steatohepatitis.

Qi Wang, Haoming Zhou, Qingfa Bu, Song Wei, Lei Li, Jinren Zhou, Shun Zhou, Wantong Su, Mu Liu, Zheng Liu, Mingming Wang, Ling Lu.

   BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is associated with the dysregulation of lipid metabolism and hepatic inflammation. The mechanism underlying NASH is unclear. We aim to investigate the role of X-box binding protein-1 (XBP1) in the progression of NASH.
METHODS: Human liver tissues obtained from patients with NASH and control group were used to assess XBP1 expression. NASH models were developed in hepatocyte-specific Xbp1 knockout (Xbp1ΔHep), macrophage specific Xbp1 knockout (Xbp1ΔMϕ), macrophage-specific Nlrp3 knockout, and wild-type (Xbp1FL/FL or Nlrp3FL/FL) mice fed with high-fat diet for 26 weeks or methionine/choline deficient diet for 6 weeks.
RESULTS: The expression of XBP1 was significantly upregulated in the liver samples from NASH patients. Hepatocyte-specific Xbp1 deficiency inhibited the development of steatohepatitis in the mice fed with the high-fat or methionine/choline deficient diets. Meanwhile, macrophage specific Xbp1 knockout mice developed less severe steatohepatitis and fibrosis than wild-type Xbp1FL/FL mice in response to the high-fat or methionine/choline deficient diets. Macrophage-specific Xbp1 knockout mice showed M2 anti-inflammatory polarization. Xbp1 deleted macrophages reduced steatohepatitis through decreased expression of NLRP3 and secretion of pro-inflammatory cytokines, which mediate M2 macrophage polarization in macrophage-specific Xbp1 knockout mice. Steatohepatitis was less severe in macrophage-specific Nlrp3 knockout mice than in wild-type Nlrp3FL/FL mice. Xbp1 deleted macrophages prevented the hepatic stellate cells activation through decreased expression of TGF-β1. Less fibrotic changes were observed in macrophage-specific Xbp1 knockout mice than in the wild-type Xbp1FL/FL mice. Inhibition of XBP1 suppressed the development of NASH.
CONCLUSION: XBP1 regulates the development of NASH. XBP1 inhibitors protect against steatohepatitis. XBP1 thus is a potential target for the treatment of NASH.
LAY SUMMARY: XBP1 is a distinct basic-region leucine zipper transcription factor whose dynamic form is dominated by splicing response upon breakdown by homeostasis in the endoplasmic reticulum and activation of the unfolded protein response. Our study demonstrated that XBP1 is upregulated in liver tissues of patients with NASH. Conditional knockout of Xbp1 in the hepatocytes resulted in decreased lipid accumulation in mice. Genetic specific deletion of Xbp1 in macrophages ameliorated nutritional steatohepatitis and fibrosis in mice by reducing the secretion of pro-inflammatory cytokine, increasing M2 macrophage polarization, and decreasing TGF-β1 expression. Pharmacological inhibition of XBP1 protects against steatohepatitis and fibrosis, highlighting a promising strategy for NASH therapy.

Keywords:  XBP1; hepatic stellate cells; hepatocytes; macrophages; non-alcoholic steatohepatitis

DOI:  https://doi.org/10.1016/j.jhep.2022.02.031
FEBS J. 2022 Mar 19.

Hsp104 N-terminal Domain Interaction with Substrates Plays a Regulatory Role in Protein Disaggregation.

Anna Harari, Guy Zoltsman, Tal Levin, Rina Rosenzweig.

  Hsp104 protein disaggregases are powerful molecular machines that harness the energy derived from ATP binding and hydrolysis to disaggregate a wide range of protein aggregates and amyloids, as well as to assist in yeast prion propagation. Little is known, however, about how Hsp104 chaperones recognize such a diversity of substrates, or indeed the contribution of the substrate-binding N-terminal domain (NTD) to Hsp104 function. Herein we present a Nuclear Magnetic Resonance (NMR) spectroscopy study which structurally characterizes the Hsp104 NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded, misfolded, amyloid, and prion substrates of Hsp104. In addition, we find that the NTD itself has chaperoning activities which help to protect the exposed hydrophobic regions of its substrates from further misfolding and aggregation, thereby priming them for threading through the Hsp104 central channel. We further demonstrate that mutations to this substrate binding groove abolish Hsp104 activation by client proteins and keep the chaperone in a partially inhibited state. The Hsp104 variant with these mutations also exhibited significantly reduced disaggregation activity and cell survival at extreme temperatures. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the N-terminal domain in protein disaggregation and yeast thermotolerance.

Keywords:  Hsp104; Molecular chaperones; NMR spectroscopy; protein disaggregation

DOI:  https://doi.org/10.1111/febs.16441
J Cell Biol. 2022 Apr 04. pii: e202107114. [Epub ahead of print]221(4):

Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration.

Emily D Duncan, Ke-Jun Han, Margaret A Trout, Rytis Prekeris.

Cell migration is a complex process that involves coordinated changes in membrane transport and actin cytoskeleton dynamics. Ras-like small monomeric GTPases, such as Rap2, play a key role in regulating actin cytoskeleton dynamics and cell adhesions. However, how Rap2 function, localization, and activation are regulated during cell migration is not fully understood. We previously identified the small GTPase Rab40b as a regulator of breast cancer cell migration. Rab40b contains a suppressor of cytokine signaling (SOCS) box, which facilitates binding to Cullin5, a known E3 ubiquitin ligase component responsible for protein ubiquitylation. In this study, we show that the Rab40b/Cullin5 complex ubiquitylates Rap2. Importantly, we demonstrate that ubiquitylation regulates Rap2 activation as well as recycling of Rap2 from the endolysosomal compartment to the lamellipodia of migrating breast cancer cells. Based on these data, we propose that Rab40b/Cullin5 ubiquitylates and regulates Rap2-dependent actin dynamics at the leading edge, a process that is required for breast cancer cell migration and invasion.

DOI: https://doi.org/10.1083/jcb.202107114
mBio. 2022 Mar 15. e0325121

Copper Induces Protein Aggregation, a Toxic Process Compensated by Molecular Chaperones.

Lisa Zuily, Nora Lahrach, Rosi Fassler, Olivier Genest, Peter Faller, Olivier Sénèque, Yann Denis, Marie-Pierre Castanié-Cornet, Pierre Genevaux, Ursula Jakob, Dana Reichmann, Marie-Thérèse Giudici-Orticoni, Marianne Ilbert.

  Copper is well known for its antimicrobial and antiviral properties. Under aerobic conditions, copper toxicity relies in part on the production of reactive oxygen species (ROS), especially in the periplasmic compartment. However, copper is significantly more toxic under anaerobic conditions, in which ROS cannot be produced. This toxicity has been proposed to arise from the inactivation of proteins through mismetallations. Here, using the bacterium Escherichia coli, we discovered that copper treatment under anaerobic conditions leads to a significant increase in protein aggregation. In vitro experiments using E. coli lysates and tightly controlled redox conditions confirmed that treatment with Cu+ under anaerobic conditions leads to severe ROS-independent protein aggregation. Proteomic analysis of aggregated proteins revealed an enrichment of cysteine- and histidine-containing proteins in the Cu+-treated samples, suggesting that nonspecific interactions of Cu+ with these residues are likely responsible for the observed protein aggregation. In addition, E. coli strains lacking the cytosolic chaperone DnaK or trigger factor are highly sensitive to copper stress. These results reveal that bacteria rely on these chaperone systems to protect themselves against Cu-mediated protein aggregation and further support our finding that Cu toxicity is related to Cu-induced protein aggregation. Overall, our work provides new insights into the mechanism of Cu toxicity and the defense mechanisms that bacteria employ to survive. IMPORTANCE With the increase of antibiotic drug resistance, alternative antibacterial treatment strategies are needed. Copper is a well-known antimicrobial and antiviral agent; however, the underlying molecular mechanisms by which copper causes cell death are not yet fully understood. Herein, we report the finding that Cu+, the physiologically relevant copper species in bacteria, causes widespread protein aggregation. We demonstrate that the molecular chaperones DnaK and trigger factor protect bacteria against Cu-induced cell death, highlighting, for the first time, the central role of these chaperones under Cu+ stress. Our studies reveal Cu-induced protein aggregation to be a central mechanism of Cu toxicity, a finding that will serve to guide future mechanistic studies and drug development.

Keywords:  DnaK; Escherichia coli; copper homeostasis; copper stress; copper tolerance; heat shock; molecular chaperone; protein aggregation; proteostasis; stress response; trigger factor

DOI:  https://doi.org/10.1128/mbio.03251-21
Nat Commun. 2022 Mar 15. 13(1): 1351

Mapping the sequence specificity of heterotypic amyloid interactions enables the identification of aggregation modifiers.

Nikolaos Louros, Meine Ramakers, Emiel Michiels, Katerina Konstantoulea, Chiara Morelli, Teresa Garcia, Nele Moonen, Sam D'Haeyer, Vera Goossens, Dietmar Rudolf Thal, Dominique Audenaert, Frederic Rousseau, Joost Schymkowitz.

Heterotypic amyloid interactions between related protein sequences have been observed in functional and disease amyloids. While sequence homology seems to favour heterotypic amyloid interactions, we have no systematic understanding of the structural rules determining such interactions nor whether they inhibit or facilitate amyloid assembly. Using structure-based thermodynamic calculations and extensive experimental validation, we performed a comprehensive exploration of the defining role of sequence promiscuity in amyloid interactions. Using tau as a model system we demonstrate that proteins with local sequence homology to tau amyloid nucleating regions can modify fibril nucleation, morphology, assembly and spreading of aggregates in cultured cells. Depending on the type of mutation such interactions inhibit or promote aggregation in a manner that can be predicted from structure. We find that these heterotypic amyloid interactions can result in the subcellular mis-localisation of these proteins. Moreover, equilibrium studies indicate that the critical concentration of aggregation is altered by heterotypic interactions. Our findings suggest a structural mechanism by which the proteomic background can modulate the aggregation propensity of amyloidogenic proteins and we discuss how such sequence-specific proteostatic perturbations could contribute to the selective cellular susceptibility of amyloid disease progression.

DOI: https://doi.org/10.1038/s41467-022-28955-9
Proc Natl Acad Sci U S A. 2022 Mar 22. 119(12): e2100670119

Lysosomal inhibition sensitizes TMEM16A-expressing cancer cells to chemotherapy.

Avani Vyas, Roberto Gomez-Casal, Silvia Cruz-Rangel, Hugo Villanueva, Andrew G Sikora, Pavithra Rajagopalan, Devraj Basu, Jonathan Pacheco, Gerald R V Hammond, Kirill Kiselyov, Umamaheswar Duvvuri.

  SignificanceCisplatin is the first line therapy for patients with head and neck cancer. However, resistance to cisplatin remains a major concern. High expression of the calcium-activated chloride channel TMEM16A in tumors portends poor survival in these patients, possibly because of drug resistance. Here, we show that TMEM16A drives the sequestration of cisplatin into lysosomes. Subsequently, cisplatin is expelled via the delivery of lysosomes to the cell surface. We show that TMEM16A enhances this process, thereby promoting cisplatin resistance. We also show that lysosomal inhibition synergizes with cisplatin to induce tumor cell death. Our data uncovers a new fundamental feature of both lysosomal physiology and cancer cell biology that can potentially impact the treatment of patients with head and neck cancer.

Keywords:  MITF; TMEM16A; cisplatin; hydroxychloroquine; lysosomal flux

DOI:  https://doi.org/10.1073/pnas.2100670119
Cell. 2022 Mar 09. pii: S0092-8674(22)00196-9. [Epub ahead of print]

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding.

Matthew Thomas Doyle, John R Jimah, Tyrone Dowdy, Shannon I Ohlemacher, Mioara Larion, Jenny E Hinshaw, Harris D Bernstein.

  Transmembrane β barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the β barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "β signal" motif of EspP to correctly orient β strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated β sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that β sheets progressively fold toward BamA to form a β barrel. Along with in vivo experiments that tracked β barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate β barrel folding.

Keywords:  BAM; membrane dynamics; membrane protein folding; outer membrane protein; β barrel

DOI:  https://doi.org/10.1016/j.cell.2022.02.016
Cancer Res. 2022 Mar 18. pii: canres.2106.2021. [Epub ahead of print]

M6A RNA Methylation Regulates Histone Ubiquitination to Support Cancer Growth and Progression.

Pooja Yadav, Panneerdoss Subbarayalu, Daisy Medina, Saif Nirzhor, Santosh Timilsina, Subapriya Rajamanickam, Vijay K Eedunuri, Yogesh Gupta, Siyuan Zheng, Nourhan Abdelfattah, Yufei Huang, Ratna Vadlamudi, Robert Hromas, Paul Meltzer, Peter Houghton, Yidong Chen, Manjeet K Rao.

Osteosarcoma is the most common malignancy of the bone, yet the survival for osteosarcoma patients is virtually unchanged over the past 30 years. This is principally because development of new therapies is hampered by a lack of recurrent mutations that can be targeted in osteosarcoma. Here, we report that epigenetic changes via mRNA methylation holds great promise to better understand the mechanisms of osteosarcoma growth and to develop targeted therapeutics. In osteosarcoma patients, the RNA demethylase ALKBH5 was amplified and higher expression correlated with copy number changes. ALKBH5 was critical for promoting osteosarcoma growth and metastasis, yet it was dispensable for normal cell survival. Me-RIP-seq analysis and functional studies showed that ALKBH5 mediates its pro-tumorigenic function by regulating m6A levels of histone deubiquitinase USP22 and the ubiquitin ligase RNF40. ALKBH5-mediated m6A deficiency in osteosarcoma led to increased expression of USP22 and RNF40 that resulted in inhibition of histone H2A monoubiquitination and induction of key pro-tumorigenic genes, consequently driving unchecked cell cycle progression, incessant replication and DNA repair. RNF40, which is historically known to ubiquitinate H2B, inhibited H2A ubiquitination in cancer by interacting with and affecting the stability of DDB1-CUL4-based ubiquitin E3 ligase complex. Taken together, this study directly links increased activity of ALKBH5 with dysregulation of USP22/RNF40 and histone ubiquitination in cancers. More broadly, these results suggest that m6A RNA methylation works in concert with other epigenetic mechanisms to control cancer growth.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2106