bims-proteo 2022-08-14 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022–08–14
35 papers selected by
Eric Chevet, INSERM

TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.
Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation.
Endoplasmic Reticulum-Associated Protein Degradation.
Mechanism of Protein Translocation by the Sec61 Translocon Complex.
The Role of the Rhomboid Superfamily in ER Protein Quality Control: From Mechanisms and Functions to Diseases.
Sorting and Export of Proteins at the Endoplasmic Reticulum.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1.
Evolutionary Aspects of the Unfolded Protein Response.
Post-Translational Regulation of HMG CoA Reductase.
ER-Phagy: Quality and Quantity Control of the Endoplasmic Reticulum by Autophagy.
Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response.
Chaperone Requirements for De Novo Folding of Saccharomyces cerevisiae Septins.
A Pipeline for Natural Small Molecule Inhibitors of Endoplasmic Reticulum Stress.
The endoplasmic reticulum membrane complex promotes proteostasis of GABAA receptors.
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.
The deubiquitinating enzymes UBP12 and UBP13 positively regulate recovery after carbon starvation by modulating BES1 stability in Arabidopsis thaliana.
The Cys/N-degron pathway in the ubiquitin-proteasome system and autophagy.
Molecular basis of MHC I quality control in the peptide loading complex.
Reciprocal interplay between OTULIN-LUBAC determines genotoxic and inflammatory NF-κB signal responses.
Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition.
A genetic model for in vivo proximity labelling of the mammalian secretome.
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription.
C11orf94/Frey is a key regulator for male fertility by controlling Izumo1 complex assembly.
Adaptation to chronic ER stress enforces pancreatic β-cell plasticity.
OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways.
TMEM164 is a new determinant of autophagy-dependent ferroptosis.
CRISPR-RNAa: targeted activation of translation using dCas13 fusions to translation initiation factors.
Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT.
Experimental and computational workflow for the analysis of tRNA pools from eukaryotic cells by mim-tRNAseq.
Uncovering the universality of self-replication in protein aggregation and its link to disease.
Comprehensive Transcriptomic Analysis of Novel Class I HDAC Proteolysis Targeting Chimeras (PROTACs).
Targeting RNA structures with small molecules.
QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1α RNase.
Seipin concentrates distinct neutral lipids via interactions with their acyl chain carboxyl esters.
uORF-Mediated Translational Regulation of ATF4 Serves as an Evolutionarily Conserved Mechanism Contributing to Non-Small-Cell Lung Cancer (NSCLC) and Stress Response.

Mol Cell. 2022 Aug 10. pii: S1097-2765(22)00663-3. [Epub ahead of print]

TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.

Linhan Wang, Jiqiang Li, Qingchen Wang, Man-Xi Ge, Jia Ji, Di Liu, Zhiyuan Wang, Yang Cao, Yaoyang Zhang, Zai-Rong Zhang.

  Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.

Keywords:  ERAD; TMUB1; endoplasmic-reticulum-associated degradation; escortase; membrane protein extraction; membrane protein quality control; p97/VCP; protein degradation; retrotranslocation

DOI:  https://doi.org/10.1016/j.molcel.2022.07.006
Cell Rep. 2022 Aug 09. pii: S2211-1247(22)00988-3. [Epub ahead of print]40(6): 111175

Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation.

Josephine Bock, Nathalie Kühnle, Julia D Knopf, Nina Landscheidt, Jin-Gu Lee, Yihong Ye, Marius K Lemberg.

  Protein degradation is fundamentally important to ensure cell homeostasis. In the endoplasmic reticulum (ER), the ER-associated degradation (ERAD) pathway targets incorrectly folded and unassembled proteins for turnover by the cytoplasmic proteasome. Previously, we showed that the rhomboid protease RHBDL4, together with p97, mediates membrane protein degradation. However, whether RHBDL4 acts in concert with additional ERAD components is unclear, and its full substrate spectrum remains to be defined. Here, we show that, in addition to membrane proteins, RHBDL4 cleaves aggregation-prone luminal ERAD substrates. Since mutations of the RHBDL4 rhomboid domain led to stabilization of substrates at the cytoplasmic side, we hypothesize that, analogous to the homolog ERAD factor derlin, RHBDL4 is directly involved in substrate retrotranslocation. RHBDL4's interaction with the erlin ERAD complex and reciprocal interaction of rhomboid substrates with erlins suggest that RHBDL4 and erlins form a complex that clips substrates and thereby rescues aggregation-prone peptides in the ER from aggregation.

Keywords:  CP: Cell biology; CP: Molecular biology; ER-associated protein degradation; intramembrane proteolysis; prohibitin family proteins Erlin1 and Erlin2; protein aggregates; rhomboid family protein

DOI:  https://doi.org/10.1016/j.celrep.2022.111175
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041247. [Epub ahead of print]

Endoplasmic Reticulum-Associated Protein Degradation.

Logesvaran Krshnan, Michael L van de Weijer, Pedro Carvalho.

Misfolded, potentially toxic proteins in the lumen and membrane of the endoplasmic reticulum (ER) are eliminated by proteasomes in the cytosol through ER-associated degradation (ERAD). The ERAD process involves the recognition of substrates in the lumen and membrane of the ER, their translocation into the cytosol, ubiquitination, and delivery to the proteasome for degradation. These ERAD steps are performed by membrane-embedded ubiquitin-ligase complexes of different specificity that together cover a wide range of substrates. Besides misfolded proteins, ERAD further contributes to quality control by targeting unassembled and mislocalized proteins. ERAD also targets a restricted set of folded proteins to influence critical ER functions such as sterol biosynthesis, calcium homeostasis, or ER contacts with other organelles. This review describes the ubiquitin-ligase complexes and the principles guiding protein degradation by ERAD.

DOI: https://doi.org/10.1101/cshperspect.a041247
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041250. [Epub ahead of print]

Mechanism of Protein Translocation by the Sec61 Translocon Complex.

Samuel Itskanov, Eunyong Park.

The endoplasmic reticulum (ER) is a major site for protein synthesis, folding, and maturation in eukaryotic cells, responsible for production of secretory proteins and most integral membrane proteins. The universally conserved protein-conducting channel Sec61 complex mediates core steps in these processes by translocating hydrophilic polypeptide segments of client proteins across the ER membrane and integrating hydrophobic transmembrane segments into the membrane. The Sec61 complex associates with several other molecular machines and enzymes to enable substrate engagement with the channel and coordination of protein translocation with translation, protein folding, and/or post-translational modifications. Recent cryo-electron microscopy and functional studies of these translocon complexes have greatly advanced our mechanistic understanding of Sec61-dependent protein biogenesis at the ER. Here, we will review the current models for how the Sec61 channel performs its functions in coordination with partner complexes.

DOI: https://doi.org/10.1101/cshperspect.a041250
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041248. [Epub ahead of print]

The Role of the Rhomboid Superfamily in ER Protein Quality Control: From Mechanisms and Functions to Diseases.

Satarupa Bhaduri, Nicola A Scott, Sonya E Neal.

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells and is a major site for protein folding, modification, and lipid synthesis. Perturbations within the ER, such as protein misfolding and high demand for protein folding, lead to dysregulation of the ER protein quality control network and ER stress. Recently, the rhomboid superfamily has emerged as a critical player in ER protein quality control because it has diverse cellular functions, including ER-associated degradation (ERAD), endosome Golgi-associated degradation (EGAD), and ER preemptive quality control (ERpQC). This breadth of function both illustrates the importance of the rhomboid superfamily in health and diseases and emphasizes the necessity of understanding their mechanisms of action. Because dysregulation of rhomboid proteins has been implicated in various diseases, such as neurological disorders and cancers, they represent promising potential therapeutic drug targets. This review provides a comprehensive account of the various roles of rhomboid proteins in the context of ER protein quality control and discusses their significance in health and disease.

DOI: https://doi.org/10.1101/cshperspect.a041248
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041258. [Epub ahead of print]

Sorting and Export of Proteins at the Endoplasmic Reticulum.

Ishier Raote, Sonashree Saxena, Vivek Malhotra.

Secretory proteins are transported from the endoplasmic reticulum (ER) to the Golgi complex in carriers that are formed by the concerted activities of cytoplasmic proteins in the coat protein complex II (COPII). COPII was first described in Saccharomyces cerevisiae and its basic functions are largely conserved throughout eukaryotes. The discovery of the TANGO1 (transport and Golgi organization 1) family of proteins is revealing insights into how cells can adapt COPII proteins to reorganize the ER exit site for the export of the most abundant and bulky molecules, collagens.

DOI: https://doi.org/10.1101/cshperspect.a041258
EMBO J. 2022 Aug 08. e110596

Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1.

Santosh Phuyal, Elena Djaerff, Anabel-Lise Le Roux, Martin J Baker, Daniela Fankhauser, Sayyed Jalil Mahdizadeh, Veronika Reiterer, Amirabbas Parizadeh, Edward Felder, Jennifer C Kahlhofer, David Teis, Marcelo G Kazanietz, Stephan Geley, Leif Eriksson, Pere Roca-Cusachs, Hesso Farhan.

  Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

Keywords:  COPII; endoplasmic reticulum; mechanobiology

DOI:  https://doi.org/10.15252/embj.2022110596
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041262. [Epub ahead of print]

Evolutionary Aspects of the Unfolded Protein Response.

Kazutoshi Mori.

The unfolded protein response (UPR) is activated when unfolded proteins accumulate in the endoplasmic reticulum (ER). The basic mechanism of the UPR in maintaining ER homeostasis has been clarified from yeast to humans. The UPR is triggered by one or more transmembrane proteins in the ER. The number of canonical UPR sensors/transducers has increased during evolution, from one (IRE1) in yeast to three (IRE1, PERK, and ATF6) in invertebrates and five (IRE1α, IRE1β, PERK, ATF6α, and ATF6β) in vertebrates. Here, I initially describe the four major changes that have occurred during evolution: (1) advent of PERK in metazoans; (2) switch in transcription factor downstream of IRE1 in metazoans; (3) switch in regulator of ER chaperone induction in vertebrates; and (4) increase in the number of ATF6-like local factors in vertebrates. I then discuss the causes of the phenotypes of vertebrate knockout animals and refer to regulated IRE1-dependent decay of mRNAs.

DOI: https://doi.org/10.1101/cshperspect.a041262
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041253. [Epub ahead of print]

Post-Translational Regulation of HMG CoA Reductase.

Youngah Jo, Russell A DeBose-Boyd.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is an endoplasmic reticulum (ER)-localized integral membrane protein that catalyzes the rate-limiting step in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). HMGCR is subjected to strict feedback control through multiple mechanisms to ensure cells constantly produce essential nonsterol isoprenoids, but do not overaccumulate cholesterol. Here, we focus on the mechanism of feedback control of HMGCR that involves its sterol-induced ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We will also discuss the how GGpp-regulated intracellular trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) inhibits HMGCR ERAD to balance the synthesis of sterol and nonsterol isoprenoids. Finally, we will summarize various mouse models, the characterization of which establish that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMGCR and cholesterol metabolism in vivo.

DOI: https://doi.org/10.1101/cshperspect.a041253
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041256. [Epub ahead of print]

ER-Phagy: Quality and Quantity Control of the Endoplasmic Reticulum by Autophagy.

Haruka Chino, Noboru Mizushima.

The endoplasmic reticulum (ER) is the largest organelle and has multiple roles in various cellular processes such as protein secretion, lipid synthesis, calcium storage, and organelle biogenesis. The quantity and quality of this organelle are controlled by the ubiquitin-proteasome system and autophagy (termed "ER-phagy"). ER-phagy is defined as the degradation of part of the ER by the vacuole or lysosomes, and there are at least two types of ER-phagy: macro-ER-phagy and micro-ER-phagy. In macro-ER-phagy, ER fragments are enclosed by autophagosomes, which is mediated by ER-phagy receptors. In micro-ER-phagy, a portion of the ER is engulfed directly by the vacuole or lysosomes. In these two pathways, some proteins in the ER lumen can be recognized selectively and subjected to ER-phagy. This review summarizes our current knowledge of ER-phagy, focusing on its membrane dynamics, molecular mechanisms, substrate specificity, and physiological significance.

DOI: https://doi.org/10.1101/cshperspect.a041256
PLoS One. 2022 ;17(8): e0271695

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response.

Mohammed R Alzahrani, Bo-Jhih Guan, Leah L Zagore, Jing Wu, Chien-Wen Chen, Donny D Licatalosi, Kristian E Baker, Maria Hatzoglou.

Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of newly synthesized RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

DOI: https://doi.org/10.1371/journal.pone.0271695
Mol Biol Cell. 2022 Aug 10. mbcE22070262

Chaperone Requirements for De Novo Folding of Saccharomyces cerevisiae Septins.

Daniel Hassell, Ashley Denney, Emily Singer, Aleyna Benson, Andrew Roth, Julia Ceglowski, Marc Steingesser, Michael McMurray.

Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted non-native conformations and formed non-native homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble post-translationally into native complexes. One septin, Cdc12, was so dependent on co-translational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, co-translational assembly pathway in which general cytosolic chaperones act as key intermediaries.

DOI: https://doi.org/10.1091/mbc.E22-07-0262
Front Pharmacol. 2022 ;13 956154

A Pipeline for Natural Small Molecule Inhibitors of Endoplasmic Reticulum Stress.

Daniela Correia da Silva, Patrícia Valentão, Paula B Andrade, David M Pereira.

  The homeostasis of eukaryotic cells is inseverable of that of the endoplasmic reticulum (ER). The main function of this organelle is the synthesis and folding of a significant portion of cellular proteins, while it is also the major calcium reservoir of the cell. Upon unresolved ER stress, a set of stress response signaling pathways that are collectively labeled as the unfolded protein response (UPR) is activated. Prolonged or intense activation of this molecular machinery may be deleterious. It is known that compromised ER homeostasis, and consequent UPR activation, characterizes the pathogenesis of neurodegenerative diseases. In an effort to discover new small molecules capable of countering ER stress, we subjected a panel of over 100 natural molecules to a battery of assays designed to evaluate several hallmarks of ER stress. The protective potential of these compounds against ER stress was evaluated at the levels of calcium homeostasis, key gene and protein expression, and levels of protein aggregation in fibroblasts. The most promising compounds were subsequently tested in neuronal cells. This framework resulted in the identification of several bioactive molecules capable of countering ER stress and deleterious events associated to it. Delphinidin stands out as the most promising candidate against neurodegeneration. This compound significantly inhibited the expression of UPR biomarkers, and displayed a strong potential to inhibit protein aggregation in the two aforementioned cell models. Our results indicate that natural products may be a valuable resource in the development of an effective therapeutic strategy against ER stress-related diseases.

Keywords:  drug discovery; endoplasmic reticulum stress; natural products; neurodegeneration; unfolded protein response

DOI:  https://doi.org/10.3389/fphar.2022.956154
iScience. 2022 Aug 19. 25(8): 104754

The endoplasmic reticulum membrane complex promotes proteostasis of GABAA receptors.

Angela L Whittsette, Ya-Juan Wang, Ting-Wei Mu.

  The endoplasmic reticulum membrane complex (EMC) plays a critical role in the biogenesis of tail-anchored proteins and a subset of multi-pass membrane proteins in the endoplasmic reticulum (ER). However, because of nearly exclusive expression of neurotransmitter-gated ion channels in the central nervous system (CNS), the role of the EMC in their biogenesis is not well understood. In this study, we demonstrated that the EMC positively regulates the surface trafficking and thus function of endogenous γ-aminobutyric acid type A (GABAA) receptors, the primary inhibitory ion channels in the mammalian brain. Moreover, among ten EMC subunits, EMC3 and EMC6 have the most prominent effect, and overexpression of EMC3 or EMC6 is sufficient to restore the function of epilepsy-associated GABAA receptor variants. In addition, EMC3 and EMC6 demonstrate endogenous interactions with major neuroreceptors, which depends on their transmembrane domains, suggesting a general role of the EMC in the biogenesis of neuroreceptors.

Keywords:  Biological sciences; Cell biology; Molecular biology; Molecular neuroscience; Neuroscience

DOI:  https://doi.org/10.1016/j.isci.2022.104754
Elife. 2022 Aug 08. pii: e78923. [Epub ahead of print]11

A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.

Willa Wen-You Yim, Hayashi Yamamoto, Noboru Mizushima.

  Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the GFP-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g., GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant RFP accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g., Halo-LC3 and Halo-GFP) are proteolytically processed to generate Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.

Keywords:  cell biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.78923
Plant Cell. 2022 Aug 09. pii: koac245. [Epub ahead of print]

The deubiquitinating enzymes UBP12 and UBP13 positively regulate recovery after carbon starvation by modulating BES1 stability in Arabidopsis thaliana.

Jiawei Xiong, Fabin Yang, Xiuhong Yao, Yuqing Zhao, Yu Wen, Honghui Lin, Hongqing Guo, Yanhai Yin, Dawei Zhang.

BRI1-EMS-SUPPRESSOR1 (BES1), a core transcription factor in the brassinosteroid (BR) signaling pathway, primarily regulates plant growth and development by influencing BR-regulated gene expression. Several E3 ubiquitin ligases regulate BES1 stability, but little is known about BES1 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain BES1 homeostasis. Here, we report that two Arabidopsis thaliana deubiquitinating enzymes, UBIQUITIN-SPECIFIC PROTEASE12 (UBP12) and UBP13, interact with BES1. UBP12 and UBP13 removed ubiquitin from polyubiquitinated BES1 to stabilize both phosphorylated and dephosphorylated forms of BES1. A double mutant, ubp12-2w ubp13-3, lacking UBP12 and UBP13 function showed both BR-deficient and BR-insensitive phenotypes, whereas transgenic plants overexpressing UBP12 or UBP13 exhibited an increased BR response. Expression of UBP12 and UPB13 was induced during recovery after carbon starvation, which led to BES1 accumulation and quick recovery of stressed plants. Our work thus establishes a mechanism by which UBP12 and UBP13 regulate BES1 protein abundance to enhance BR-regulated growth during recovery after carbon starvation.

DOI: https://doi.org/10.1093/plcell/koac245
Trends Cell Biol. 2022 Aug 06. pii: S0962-8924(22)00175-1. [Epub ahead of print]

The Cys/N-degron pathway in the ubiquitin-proteasome system and autophagy.

Ah Jung Heo, Chang Hoon Ji, Yong Tae Kwon.

  The N-degron pathway is a degradative system in which the N-terminal residues of proteins modulate the half-lives of proteins and other cellular materials. The majority of amino acids in the genetic code have the potential to induce cis or trans degradation in diverse processes, which requires selective recognition between N-degrons and cognate N-recognins. Of particular interest is the Cys/N-degron branch, in which the N-terminal cysteine (Nt-Cys) induces proteolysis via either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome pathway (ALP), depending on physiological conditions. Recent studies provided new insights into the central role of Nt-Cys in sensing the fluctuating levels of oxygen and reactive oxygen species (ROS). Here, we discuss the components, regulations, and functions of the Cys/N-degron pathway.

Keywords:  N-recognin; N-terminal oxidation; autophagy; oxygen sensor; proteolysis; the N-degron pathway

DOI:  https://doi.org/10.1016/j.tcb.2022.07.005
Nat Commun. 2022 Aug 10. 13(1): 4701

Molecular basis of MHC I quality control in the peptide loading complex.

Alexander Domnick, Christian Winter, Lukas Sušac, Leon Hennecke, Mario Hensen, Nicole Zitzmann, Simon Trowitzsch, Christoph Thomas, Robert Tampé.

Major histocompatibility complex class I (MHC I) molecules are central to adaptive immunity. Their assembly, epitope selection, and antigen presentation are controlled by the MHC I glycan through a sophisticated network of chaperones and modifying enzymes. However, the mechanistic integration of the corresponding processes remains poorly understood. Here, we determine the multi-chaperone-client interaction network of the peptide loading complex (PLC) and report the PLC editing module structure by cryogenic electron microscopy at 3.7 Å resolution. Combined with epitope-proofreading studies of the PLC in near-native lipid environment, these data show that peptide-receptive MHC I molecules are stabilized by multivalent chaperone interactions including the calreticulin-engulfed mono-glucosylated MHC I glycan, which only becomes accessible for processing by α-glucosidase II upon loading of optimal epitopes. Our work reveals allosteric coupling between peptide-MHC I assembly and glycan processing. This inter-process communication defines the onset of an adaptive immune response and provides a prototypical example of the tightly coordinated events in endoplasmic reticulum quality control.

DOI: https://doi.org/10.1038/s41467-022-32384-z
Proc Natl Acad Sci U S A. 2022 Aug 16. 119(33): e2123097119

Reciprocal interplay between OTULIN-LUBAC determines genotoxic and inflammatory NF-κB signal responses.

Mingqi Li, Ling Li, Sarah Asemota, David Kakhniashvili, Ramesh Narayanan, Xusheng Wang, Francesca-Fang Liao.

  Targeting nuclear factor-kappa B (NF-κB) represents a highly viable strategy against chemoresistance in cancers as well as cell death. Ubiquitination, including linear ubiquitination mediated by the linear ubiquitin chain assembly complex (LUBAC), is emerging as a crucial mechanism of overactivated NF-κB signaling. Ovarian tumor family deubiquitinase OTULIN is the only linear linkage-specific deubiquitinase; however, the molecular mechanisms of how it counteracts LUBAC-mediated NF-κB activation have been largely unknown. Here, we identify Lys64/66 of OTULIN for linear ubiquitination facilitated in a LUBAC-dependent manner as a necessary event required for OTULIN-LUBAC interaction under unstressed conditions, which becomes deubiquitinated by OTULIN itself in response to genotoxic stress. Furthermore, this self-deubiquitination of OTULIN occurs intermolecularly, mediated by OTULIN dimerization, resulting in the subsequent dissociation of OTULIN from the LUBAC complex and NF-κB overactivation. Oxidative stress induces OTULIN dimerization via cysteine-mediated covalent disulfide bonds. Our study reveals that the status of the physical interaction between OTULIN and LUBAC is a crucial determining factor for the genotoxic NF-κB signaling, as measured by cell survival and proliferation, while OTULIN loss of function resulting from its dimerization and deubiquitination leads to a dissociation of OTULIN from the LUBAC complex. Of note, similar molecular mechanisms apply to the inflammatory NF-κB signaling in response to tumor necrosis factor α. Hence, a fuller understanding of the detailed molecular mechanisms underlying the disruption of the OTULIN-LUBAC interaction will be instrumental for developing future therapeutic strategies against cancer chemoresistance and necroptotic processes pertinent to numerous human diseases.

Keywords:  LUBAC; NF-κB; OTULIN; deubiquitinases; inflammation

DOI:  https://doi.org/10.1073/pnas.2123097119
J Cell Biol. 2022 Sep 05. pii: e202111050. [Epub ahead of print]221(9):

Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition.

Qinqin Jiang, Martina Foglizzo, Yaroslav I Morozov, Xuejiao Yang, Arindam Datta, Lei Tian, Vaughn Thada, Weihua Li, Elton Zeqiraj, Roger A Greenberg.

The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.

DOI: https://doi.org/10.1083/jcb.202111050
Open Biol. 2022 Aug;12(8): 220149

A genetic model for in vivo proximity labelling of the mammalian secretome.

Rui Yang, Amanda S Meyer, Ilia A Droujinine, Namrata D Udeshi, Yanhui Hu, Jinjin Guo, Jill A McMahon, Dominique K Carey, Charles Xu, Qiao Fang, Jihui Sha, Shishang Qin, David Rocco, James Wohlschlegel, Alice Y Ting, Steven A Carr, Norbert Perrimon, Andrew P McMahon.

  Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.

Keywords:  BirA; TurboID; inter-organ communication; proximity-labelling; secretome; serum proteins

DOI:  https://doi.org/10.1098/rsob.220149
EMBO J. 2022 Aug 08. e109720

SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription.

Prajakta Tathe, K V S Rammohan Chowdary, Krushna Chandra Murmu, Punit Prasad, Subbareddy Maddika.

  Dynamic regulation of phosphorylation and dephosphorylation of histones is essential for eukaryotic transcription, but the enzymes engaged in histone dephosphorylation are not fully explored. Here, we show that the tyrosine phosphatase SHP-1 dephosphorylates histone H2B and plays a critical role during transition from the initiation to the elongation stage of transcription. Nuclear-localized SHP-1 is associated with the Paf1 complex at chromatin and dephosphorylates H2B at tyrosine 121. Moreover, knockout of SHP-1, or expression of a mutant mimicking constitutive phosphorylation of H2B Y121, leads to a reduction in genome-wide H2B ubiquitination, which subsequently causes defects in RNA polymerase II-dependent transcription. Mechanistically, we demonstrate that Y121 phosphorylation precludes H2B's interaction with the E2 enzyme, indicating that SHP-1-mediated dephosphorylation of this residue may be a prerequisite for efficient H2B ubiquitination. Functionally, we find that SHP-1-mediated H2B dephosphorylation contributes to maintaining basal autophagic flux in cells through the efficient transcription of autophagy and lysosomal genes. Collectively, our study reveals an important modification of histone H2B regulated by SHP-1 that has a role during eukaryotic transcription.

Keywords:  H2B ubiquitination; Paf1 complex; SHP-1; histone H2B; transcription

DOI:  https://doi.org/10.15252/embj.2021109720
Sci Adv. 2022 Aug 12. 8(32): eabo6049

C11orf94/Frey is a key regulator for male fertility by controlling Izumo1 complex assembly.

Whendy Contreras, Caroline Wiesehöfer, Dora Schreier, Nadja Leinung, Petra Peche, Gunther Wennemuth, Marc Gentzel, Bernd Schröder, Torben Mentrup.

Although gamete fusion represents the central event in sexual reproduction, the required protein machinery is poorly defined. In sperm cells, Izumo1 and several Izumo1-associated proteins play an essential role for this process. However, so far, the mechanisms underlying transport and maturation of Izumo1 and its incorporation into high molecular weight complexes are incompletely defined. Here, we provide a detailed characterization of the C11orf94 protein, which we rename Frey, which provides a platform for the assembly of Izumo1 complexes. By retaining Izumo1 in the endoplasmic reticulum, Frey facilitates its incorporation into high molecular weight complexes. To fulfill its function, the unstable Frey protein is stabilized within the catalytic center of an intramembrane protease. Loss of Frey results in reduced assembly of Izumo1 complexes and male infertility due to impaired gamete fusion. Collectively, these findings provide mechanistic insights into the early biogenesis and functional relevance of Izumo1 complexes.

DOI: https://doi.org/10.1126/sciadv.abo6049
Nat Commun. 2022 Aug 08. 13(1): 4621

Adaptation to chronic ER stress enforces pancreatic β-cell plasticity.

Chien-Wen Chen, Bo-Jhih Guan, Mohammed R Alzahrani, Zhaofeng Gao, Long Gao, Syrena Bracey, Jing Wu, Cheikh A Mbow, Raul Jobava, Leena Haataja, Ajay H Zalavadia, Ashleigh E Schaffer, Hugo Lee, Thomas LaFramboise, Ilya Bederman, Peter Arvan, Clayton E Mathews, Ivan C Gerling, Klaus H Kaestner, Boaz Tirosh, Feyza Engin, Maria Hatzoglou.

Pancreatic β-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise β-cell identity is unknown. We show here under reversible, chronic stress conditions β-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of β-cell function and identity. Upon recovery from stress, β-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while β-cells show resilience to episodic ER stress, when episodes exceed a threshold, β-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest β-cell adaptive exhaustion contributes to diabetes pathogenesis.

DOI: https://doi.org/10.1038/s41467-022-32425-7
Cell Death Dis. 2022 Aug 08. 13(8): 694

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways.

Daisuke Oikawa, Min Gi, Hidetaka Kosako, Kouhei Shimizu, Hirotaka Takahashi, Masayuki Shiota, Shuhei Hosomi, Keidai Komakura, Hideki Wanibuchi, Daisuke Tsuruta, Tatsuya Sawasaki, Fuminori Tokunaga.

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-/--mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

DOI: https://doi.org/10.1038/s41419-022-05145-5
Autophagy. 2022 Aug 10.

TMEM164 is a new determinant of autophagy-dependent ferroptosis.

Jiao Liu, Yang Liu, Yuan Wang, Changfeng Li, Yangchun Xie, Daniel J Klionsky, Rui Kang, Daolin Tang.

  Macroautophagy (hereafter "autophagy") is a membrane-mediated biological process that involves engulfing and delivering cytoplasmic components to lysosomes for degradation. In addition to autophagy's pro-survival effect during nutrient starvation, excessive activation of autophagy machinery can also cause regulated cell death, especially iron-dependent ferroptosis. Here, we report a key role for TMEM164 (transmembrane protein 164) in selectively mediating ATG5 (autophagy related 5)-dependent autophagosome formation during ferroptosis, rather than during starvation. In contrast, the membrane protein ATG9A (autophagy related 9A) is dispensable for the formation of autophagosomes during ferroptosis. TMEM164-mediated autophagy degrades ferritin, GPX4 (glutathione peroxidase 4), and lipid droplets to increase iron accumulation and lipid peroxidation, thereby promoting ferroptotic cell death. Consequently, the loss of TMEM164 limits the anticancer activity of ferroptosis-mediated cytotoxicity in mice. High TMEM164 expression is associated with improved survival and increased immune cell infiltration in patients with pancreatic cancer. These findings establish a new mode of autophagy-dependent ferroptosis.

Keywords:  Autophagy; cell death; ferroptosis; membrane protein; tumor immunity

DOI:  https://doi.org/10.1080/15548627.2022.2111635
Nucleic Acids Res. 2022 Aug 11. pii: gkac680. [Epub ahead of print]

CRISPR-RNAa: targeted activation of translation using dCas13 fusions to translation initiation factors.

Peter B Otoupal, Brady F Cress, Jennifer A Doudna, Joseph S Schoeniger.

Tools for synthetically controlling gene expression are a cornerstone of genetic engineering. CRISPRi and CRISPRa technologies have been applied extensively for programmable modulation of gene transcription, but there are few such tools for targeted modulation of protein translation rates. Here, we employ CRISPR-Cas13 as a programmable activator of translation. We develop a novel variant of the catalytically-deactivated Cas13d enzyme dCasRx by fusing it to translation initiation factor IF3. We demonstrate dCasRx-IF3's ability to enhance expression 21.3-fold above dCasRx when both are targeted to the start of the 5' untranslated region of mRNA encoding red fluorescent protein in Escherichia coli. Activation of translation is location-dependent, and we show dCasRx-IF3 represses translation when targeted to the ribosomal binding site, rather than enhancing it. We provide evidence that dCasRx-IF3 targeting enhances mRNA stability relative to dCasRx, providing mechanistic insights into how this new tool functions to enhance gene expression. We also demonstrate targeted upregulation of native LacZ 2.6-fold, showing dCasRx-IF3's ability to enhance expression of endogenous genes. dCasRx-IF3 requires no additional host modification to influence gene expression. This work outlines a novel approach, CRISPR-RNAa, for post-transcriptional control of translation to activate gene expression.

DOI: https://doi.org/10.1093/nar/gkac680
Nat Commun. 2022 Aug 10. 13(1): 4692

Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT.

S M Ayala Mariscal, M L Pigazzini, Y Richter, M Özel, I L Grothaus, J Protze, K Ziege, M Kulke, M ElBediwi, J V Vermaas, L Colombi Ciacchi, S Köppen, F Liu, J Kirstein.

Huntington's disease is a neurodegenerative disease caused by an expanded polyQ stretch within Huntingtin (HTT) that renders the protein aggregation-prone, ultimately resulting in the formation of amyloid fibrils. A trimeric chaperone complex composed of Hsc70, DNAJB1 and Apg2 can suppress and reverse the aggregation of HTTExon1Q48. DNAJB1 is the rate-limiting chaperone and we have here identified and characterized the binding interface between DNAJB1 and HTTExon1Q48. DNAJB1 exhibits a HTT binding motif (HBM) in the hinge region between C-terminal domains (CTD) I and II and binds to the polyQ-adjacent proline rich domain (PRD) of soluble as well as aggregated HTT. The PRD of HTT represents an additional binding site for chaperones. Mutation of the highly conserved H244 of the HBM of DNAJB1 completely abrogates the suppression and disaggregation of HTT fibrils by the trimeric chaperone complex. Notably, this mutation does not affect the binding and remodeling of any other protein substrate, suggesting that the HBM of DNAJB1 is a specific interaction site for HTT. Overexpression of wt DNAJB1, but not of DNAJB1H244A can prevent the accumulation of HTTExon1Q97 aggregates in HEK293 cells, thus validating the biological significance of the HBM within DNAJB1.

DOI: https://doi.org/10.1038/s41467-022-32370-5
STAR Protoc. 2022 Sep 16. 3(3): 101579

Experimental and computational workflow for the analysis of tRNA pools from eukaryotic cells by mim-tRNAseq.

Andrew Behrens, Danny D Nedialkova.

  Quantifying tRNAs is crucial for understanding how they regulate mRNA translation but is hampered by their extensive sequence similarity and premature termination of reverse transcription at multiple modified nucleotides. Here, we describe the use of modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which overcomes these limitations with optimized library construction and a comprehensive toolkit for data analysis and visualization. We outline algorithm improvements that enhance the efficiency and accuracy of read alignment and provide details on data analysis outputs using example datasets. For complete details on the use and execution of this protocol, please refer to Behrens et al. (2021).

Keywords:  Bioinformatics; Gene Expression; Molecular Biology; RNAseq; Sequencing; Systems biology

DOI:  https://doi.org/10.1016/j.xpro.2022.101579
Sci Adv. 2022 Aug 12. 8(32): eabn6831

Uncovering the universality of self-replication in protein aggregation and its link to disease.

Georg Meisl, Catherine K Xu, Jonathan D Taylor, Thomas C T Michaels, Aviad Levin, Daniel Otzen, David Klenerman, Steve Matthews, Sara Linse, Maria Andreasen, Tuomas P J Knowles.

Fibrillar protein aggregates are a hallmark of a range of human disorders, from prion diseases to dementias, but are also encountered in several functional contexts. Yet, the fundamental links between protein assembly mechanisms and their functional or pathological roles have remained elusive. Here, we analyze the aggregation kinetics of a large set of proteins that self-assemble by a nucleated-growth mechanism, from those associated with disease, over those whose aggregates fulfill functional roles in biology, to those that aggregate only under artificial conditions. We find that, essentially, all such systems, regardless of their biological role, are capable of self-replication. However, for aggregates that have evolved to fulfill a structural role, the rate of self-replication is too low to be significant on the biologically relevant time scale. By contrast, all disease-related proteins are able to self-replicate quickly compared to the time scale of the associated disease. Our findings establish the ubiquity of self-replication and point to its potential importance across aggregation-related disorders.

DOI: https://doi.org/10.1126/sciadv.abn6831
Biochemistry. 2022 Aug 10.

Comprehensive Transcriptomic Analysis of Novel Class I HDAC Proteolysis Targeting Chimeras (PROTACs).

India M Baker, Joshua P Smalley, Khadija A Sabat, James T Hodgkinson, Shaun M Cowley.

The class I histone deacetylase (HDAC) enzymes;HDAC1,2 and 3 form the catalytic engine of at least seven structurally distinct multiprotein complexes in cells. These molecular machines play a vital role in the regulation of chromatin accessibility and gene activity via the removal of acetyl moieties from lysine residues within histone tails. Their inhibition via small molecule inhibitors has beneficial effects in a number of disease types, including the clinical treatment of hematological cancers. We have previously reported a library of proteolysis targeting chimeras (PROTACs) incorporating a benzamide-based HDAC ligand (from CI-994), with an alkyl linker and ligand for the von Hippel-Lindau (VHL) E3 ubiquitin ligase that degrade HDAC1-3 at submicromolar concentrations. Here we report the addition of two novel PROTACs (JPS026 and JPS027), which utilize a ligand for the cellular inhibitor of apoptosis (IAP) family of E3 ligases. We found that both VHL (JPS004)- and IAP (JPS026)-based PROTACs degrade HDAC1-3 and induce histone acetylation to a similar degree. However, JPS026 is significantly more potent at inducing cell death in HCT116 cells than is JPS004. RNA sequencing analysis of PROTAC-treated HCT116 cells showed a distinct gene expression signature in which cell cycle and DNA replication machinery are repressed. Components of the mTORC1 and -2 complexes were also reduced, leading to an increase in FOXO3 and downstream target genes that regulate autophagy and apoptosis. In summary, a novel combination of HDAC and IAP ligands generates a PROTAC with a potent ability to stimulate apoptosis and differential gene expression in human cancer cells.

DOI: https://doi.org/10.1021/acs.biochem.2c00288
Nat Rev Drug Discov. 2022 Aug 08.

Targeting RNA structures with small molecules.

Jessica L Childs-Disney, Xueyi Yang, Quentin M R Gibaut, Yuquan Tong, Robert T Batey, Matthew D Disney.

RNA adopts 3D structures that confer varied functional roles in human biology and dysfunction in disease. Approaches to therapeutically target RNA structures with small molecules are being actively pursued, aided by key advances in the field including the development of computational tools that predict evolutionarily conserved RNA structures, as well as strategies that expand mode of action and facilitate interactions with cellular machinery. Existing RNA-targeted small molecules use a range of mechanisms including directing splicing - by acting as molecular glues with cellular proteins (such as branaplam and the FDA-approved risdiplam), inhibition of translation of undruggable proteins and deactivation of functional structures in noncoding RNAs. Here, we describe strategies to identify, validate and optimize small molecules that target the functional transcriptome, laying out a roadmap to advance these agents into the next decade.

DOI: https://doi.org/10.1038/s41573-022-00521-4
J Chem Inf Model. 2022 Aug 12.

QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1α RNase.

Sayyed Jalil Mahdizadeh, Emil Pålsson, Antonio Carlesso, Eric Chevet, Leif A Eriksson.

A range of in silico methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1α (IRE1). Using Protein-RNA molecular docking along with a series of extensive restrained/unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid-general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation.

DOI: https://doi.org/10.1021/acs.jcim.2c00735
J Cell Biol. 2022 Sep 05. pii: e202112068. [Epub ahead of print]221(9):

Seipin concentrates distinct neutral lipids via interactions with their acyl chain carboxyl esters.

Mike F Renne, Robin A Corey, Joana Veríssimo Ferreira, Phillip J Stansfeld, Pedro Carvalho.

Lipid droplets (LDs) are essential for cellular lipid homeostasis by storing diverse neutral lipids (NLs), such as triacylglycerol (TAG), steryl esters (SE), and retinyl esters (RE). A proper assembly of TAG-containing LDs at the ER requires Seipin, a conserved protein often mutated in lipodystrophies. Here, we show that the yeast Seipin Sei1 and its partner Ldb16 also promote the storage of other NL in LDs. Importantly, this role of Sei1/Ldb16 is evolutionarily conserved as expression of human-Seipin restored normal SE-containing LDs in yeast Seipin mutants. As in the case of TAG, the formation of SE-containing LDs requires interactions between hydroxyl-residues in human Seipin or yeast Ldb16 with NL carboxyl esters. These findings provide a universal mechanism for Seipin-mediated LD formation and suggest a model for how Seipin distinguishes NLs from aliphatic phospholipid acyl chains in the center of the membrane bilayer.

DOI: https://doi.org/10.1083/jcb.202112068
J Mol Evol. 2022 Aug 13.

uORF-Mediated Translational Regulation of ATF4 Serves as an Evolutionarily Conserved Mechanism Contributing to Non-Small-Cell Lung Cancer (NSCLC) and Stress Response.

Wenjing Xiao, Yang Sun, Jinpeng Xu, Na Zhang, Lina Dong.

  Diseases and environmental stresses are two distinct challenges for virtually all living organisms. In light of evolution, cellular responses to diseases and stresses might share similar molecular mechanisms, but the detailed regulation pathway is not reported yet.We obtained the transcriptomes and translatomes from several NSCLC (non-small-cell lung cancer) patients as well as from different species under normal or stress conditions. We found that the translation level of gene ATF4 is remarkably enhanced in NSCLC due to the reduced number of ribosomes binding to its upstream open reading frames (uORFs). We also showed the evolutionary conservation of this uORF-ATF4 regulation in the stress response of other species. Molecular experiments showed that knockdown of ATF4 reduced the cell growth rate while overexpression of ATF4 enhanced cell growth, especially for the ATF4 allele with mutated uORFs. Population genetics analyses in multiple species verified that the mutations that abolish uATGs (start codon of uORFs) are highly deleterious, suggesting the functional importance of uORFs.Our study proposes an evolutionarily conserved pattern that enhances the ATF4 translation by uORFs upon stress or disease. We generalized the concept of cellular response to diseases and stresses. These two biological processes may share similar molecular mechanisms.

Keywords:  ATF4; Evolutionarily conserved; Non-small-cell lung cancer (NSCLC); Stress response; Translation regulation; Upstream open reading frame (uORF)

DOI:  https://doi.org/10.1007/s00239-022-10068-y