bims-proteo 2022-12-11 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022–12–11
25 papers selected by
Eric Chevet, INSERM

Selective ribosome profiling reveals a role for SecB in the co-translational inner membrane protein biogenesis.
ER-associated RNA silencing promotes ER quality control.
Regulation of autophagosome biogenesis and mitochondrial bioenergetics by the cholesterol transport protein GRAMD1C.
BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1.
Is bRaQCing bad? New roles for ribosome associated quality control factors in stress granule regulation.
Conserved degronome features governing quality control associated proteolysis.
CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response.
Machine Learning Modeling of Protein-intrinsic Features Predicts Tractability of Targeted Protein Degradation.
USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction.
Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity.
DAP5 enables main ORF translation on mRNAs with structured and uORF-containing 5' leaders.
The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis.
Substitution of PINK1 Gly411 modulates substrate receptivity and turnover.
Arginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate.
Translation-complex profiling of fission yeast cells reveals dynamic rearrangements of scanning ribosomal subunits upon nutritional stress.
Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation.
Targeted Protein Degradation: Design Considerations for PROTAC Development.
Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT.
Multiple sub-state structures of SERCA2b reveal conformational overlap at transition steps during the catalytic cycle.
Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma.
The IRE1α-XBP1 Signaling Axis Promotes Glycolytic Reprogramming in Response to Inflammatory Stimuli.
Proximity labeling technologies to illuminate glycan-protein interactions.
Regulation of ribonucleoprotein condensates by RNase L during viral infection.
Protein phosphatase 1 subunit PPP1R14B stabilizes STMN1 to promote progression and paclitaxel resistance in triple-negative breast cancer.
Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Cell Rep. 2022 Dec 06. pii: S2211-1247(22)01659-X. [Epub ahead of print]41(10): 111776

Selective ribosome profiling reveals a role for SecB in the co-translational inner membrane protein biogenesis.

Lena Eismann, Igor Fijalkowski, Carla Verónica Galmozzi, Jiří Koubek, Frank Tippmann, Petra Van Damme, Günter Kramer.

  The chaperone SecB has been implicated in de novo protein folding and translocation across the membrane, but it remains unclear which nascent polypeptides SecB binds, when during translation SecB acts, how SecB function is coordinated with other chaperones and targeting factors, and how polypeptide engagement contributes to protein biogenesis. Using selective ribosome profiling, we show that SecB binds many nascent cytoplasmic and translocated proteins generally late during translation and controlled by the chaperone trigger factor. Revealing an uncharted role in co-translational translocation, inner membrane proteins (IMPs) are the most prominent nascent SecB interactors. Unlike other substrates, IMPs are bound early during translation, following the membrane targeting by the signal recognition particle. SecB remains bound until translation is terminated, and contributes to membrane insertion. Our study establishes a role of SecB in the co-translational maturation of proteins from all cellular compartments and functionally implicates cytosolic chaperones in membrane protein biogenesis.

Keywords:  CP: Molecular biology; SecB; chaperone; compartment-specific ribosome profiling; membrane insertion; proteostasis; selective ribosome profiling; signal recognition particle; translocation; trigger factor

DOI:  https://doi.org/10.1016/j.celrep.2022.111776
Nat Cell Biol. 2022 Dec;24(12): 1714-1725

ER-associated RNA silencing promotes ER quality control.

Sotirios Efstathiou, Franziska Ottens, Lena-Sophie Schütter, Sonia Ravanelli, Nikolaos Charmpilas, Aljona Gutschmidt, Jérémie Le Pen, Niels H Gehring, Eric A Miska, Jorge Bouças, Thorsten Hoppe.

The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.

DOI: https://doi.org/10.1038/s41556-022-01025-4
Autophagy. 2022 Dec 05.

Regulation of autophagosome biogenesis and mitochondrial bioenergetics by the cholesterol transport protein GRAMD1C.

Chara Charsou, Matthew Yoke Wui Ng, Anne Simonsen.

  Autophagosomes are crucial components of the cellular recycling machinery that form at endoplasmic reticulum (ER)-associated sites. As the autophagosome membrane is largely devoid of transmembrane proteins, autophagosome biogenesis is thought to be largely regulated by lipid transfer and lipid modifications, as well as membrane-associated proteins. While the membrane origin of autophagosomes and their lipid composition are still incompletely understood, previous studies have found the autophagosome membrane to be enriched in unsaturated fatty acids and have little cholesterol, suggesting that cholesterol removal is an integral step during autophagosome biogenesis. In our study, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and identify the ER-localized cholesterol transport protein GRAMD1C as a negative regulator of starvation-induced macroautophagy/autophagy.

Keywords:  Aster; GRAMD1C; VASt; autophagy; ccRCC; cholesterol

DOI:  https://doi.org/10.1080/15548627.2022.2155020
iScience. 2022 Dec 22. 25(12): 105626

BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1.

Robert Hromas, Gayathri Srinivasan, Ming Yang, Aruna Jaiswal, Taylor A Totterdale, Linda Phillips, Austin Kirby, Nazli Khodayari, Mark Brantley, Elizabeth A Williamson, Kimi Y Kong.

  Tumors with BRCA1 mutations have poor prognoses due to genomic instability. Yet this genomic instability has risks and BRCA1-deficient (def) cancer cells must develop pathways to mitigate these risks. One such risk is the accumulation of unfolded proteins in BRCA1-def cancers from increased mutations due to their loss of genomic integrity. Little is known about how BRCA1-def cancers survive their genomic instability. Here we show that BRCA1 is an E3 ligase in the endoplasmic reticulum (ER) that targets the unfolded protein response (UPR) stress sensors, Eukaryotic Translation Initiation Factor 2-alpha Kinase 3 (PERK) and Serine/Threonine-Protein Kinase/Endoribonuclease Inositol-Requiring Enzyme 1 (IRE1) for ubiquitination and subsequent proteasome-mediated degradation. When BRCA1 is mutated or depleted, both PERK and IRE1 protein levels are increased, resulting in a constitutively activated UPR. Furthermore, the inhibition of protein folding or UPR signaling markedly decreases the overall survival of BRCA1-def cancer cells. Our findings define a mechanism used by the BRCA1-def cancer cells to survive their increased unfolded protein burden which can be used to develop new therapeutic strategies to treat these cancers.

Keywords:  Cancer; Cell biology; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2022.105626
Biochem Soc Trans. 2022 Dec 09. pii: BST20220549. [Epub ahead of print]

Is bRaQCing bad? New roles for ribosome associated quality control factors in stress granule regulation.

Noah S Helton, Stephanie L Moon.

  Maintenance of proteostasis is of utmost importance to cellular viability and relies on the coordination of many post-transcriptional processes to respond to stressful stimuli. Stress granules (SGs) are RNA-protein condensates that form after translation initiation is inhibited, such as during the integrated stress response (ISR), and may facilitate cellular adaptation to stress. The ribosome-associated quality control (RQC) pathway is a critical translation monitoring system that recognizes aberrant mRNAs encoding potentially toxic nascent peptides to target them for degradation. Both SG regulation and the RQC pathway are directly associated with translation regulation, thus it is of no surprise recent developments have demonstrated a connection between them. VCP's function in the stress activated RQC pathway, ribosome collisions activating the ISR, and the regulation of the 40S ribosomal subunit by canonical SG proteins during the RQC all connect SGs to the RQC pathway. Because mutations in genes that are involved in both SG and RQC regulation are associated with degenerative and neurological diseases, understanding the coordination and interregulation of SGs and RQC may shed light on disease mechanisms. This minireview will highlight recent advances in understanding how SGs and the RQC pathway interact in health and disease contexts.

Keywords:  integrated stress response; proteostasis; ribosome-associated quality control; ribosomes; stress granules; translation

DOI:  https://doi.org/10.1042/BST20220549
Nat Commun. 2022 Dec 08. 13(1): 7588

Conserved degronome features governing quality control associated proteolysis.

Bayan Mashahreh, Shir Armony, Kristoffer Enøe Johansson, Alon Chappleboim, Nir Friedman, Richard G Gardner, Rasmus Hartmann-Petersen, Kresten Lindorff-Larsen, Tommer Ravid.

The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identify a large cohort of proteome-derived degrons, some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm establishes constraints governing degron potency, including the amino acid composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons are present in viral and human proteins, suggesting conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of protein half-life.

DOI: https://doi.org/10.1038/s41467-022-35298-y
Leukemia. 2022 Dec 06.

CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response.

Jonas S Jutzi, Anna E Marneth, María José Jiménez-Santos, Jessica Hem, Angel Guerra-Moreno, Benjamin Rolles, Shruti Bhatt, Samuel A Myers, Steven A Carr, Yuning Hong, Olga Pozdnyakova, Peter van Galen, Fátima Al-Shahrour, Anna S Nam, Ann Mullally.

Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

DOI: https://doi.org/10.1038/s41375-022-01781-0
Genomics Proteomics Bioinformatics. 2022 Dec 06. pii: S1672-0229(22)00149-8. [Epub ahead of print]

Machine Learning Modeling of Protein-intrinsic Features Predicts Tractability of Targeted Protein Degradation.

Wubing Zhang, Shourya S Roy Burman, Jiaye Chen, Katherine A Donovan, Yang Cao, Chelsea Shu, Boning Zhang, Zexian Zeng, Shengqing Gu, Yi Zhang, Dian Li, Eric S Fischer, Collin Tokheim, X Shirley Liu.

  Targeted protein degradation (TPD) has rapidly emerged as a therapeutic modality to eliminate previously undruggable proteins by repurposing the cell's endogenous protein degradation machinery. However, the susceptibility of proteins for targeting by TPD approaches, termed "degradability", is largely unknown. Here, we developed a machine learning model, model-free analysis of protein degradability (MAPD), to predict degradability from features intrinsic to protein targets. MAPD shows accurate performance in predicting kinases that are degradable by TPD compounds [with an area under precision recall curve (AUPRC) of 0.759 and area under the receiver operating characteristic curve (AUROC) of 0.775] and is likely generalizable to independent non-kinase proteins. We found five features with statistical significance to achieve optimal prediction, with ubiquitination potential being the most predictive. By structural modeling, we found that E2-accessible ubiquitination sites, but not lysine residues in general, are particularly associated with kinase degradability. Finally, we extended MAPD predictions to the entire proteome to find 964 disease-causing proteins, including 278 cancer genes, that may be tractable to TPD drug development. Running title: Zhang W et al / Protein-intrinsic Features Predict Degradability.

Keywords:  Degradability; Machine learning; Protein-intrinsic features; Targeted protein degradation; Ubiquitination

DOI:  https://doi.org/10.1016/j.gpb.2022.11.008
Cell Rep. 2022 Dec 06. pii: S2211-1247(22)01524-8. [Epub ahead of print]41(10): 111653

USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction.

Alexandra Hertel, Ludovico Martins Alves, Henrik Dutz, Georg Tascher, Florian Bonn, Manuel Kaulich, Ivan Dikic, Stefan Eimer, Florian Steinberg, Anja Bremm.

  The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.

Keywords:  CP: Cell biology; LAMTOR1; Rab7; Ragulator complex; USP32; autophagy; deubiquitinase (DUB); mTORC1; ubiquitin; v-ATPase

DOI:  https://doi.org/10.1016/j.celrep.2022.111653
Cell Rep. 2022 Dec 06. pii: S2211-1247(22)01658-8. [Epub ahead of print]41(10): 111775

Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity.

Joseph E Kaserman, Rhiannon B Werder, Feiya Wang, Taylor Matte, Michelle I Higgins, Mark Dodge, Jonathan Lindstrom-Vautrin, Pushpinder Bawa, Anne Hinds, Esther Bullitt, Ignacio S Caballero, Xu Shi, Robert E Gerszten, Nicola Brunetti-Pierri, Marc Liesa, Carlos Villacorta-Martin, Anthony N Hollenberg, Darrell N Kotton, Andrew A Wilson.

  Individuals homozygous for the "Z" mutation in alpha-1 antitrypsin deficiency are known to be at increased risk for liver disease. It has also become clear that some degree of risk is similarly conferred by the heterozygous state. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of a single Z alpha-1 antitrypsin (ZAAT) allele on hepatocyte biology. Here, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-hepatocytes (iHeps). We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations including altered endoplasmic reticulum (ER) and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in cell subpopulations. Our model of MZ heterozygosity thus provides evidence that a single Z allele is sufficient to disrupt hepatocyte homeostatic function.

Keywords:  CP: Metabolism; CP: Stem cell research; ER stress; alpha-1 antitrypsin deficiency; cellular heterogeneity; iPSC-derived hepatocytes; induced pluripotent stem cells; liver fibrosis; metabolic dysregulation; mitochondrial dysfunction; proteostasis; unfolded protein response

DOI:  https://doi.org/10.1016/j.celrep.2022.111775
Nat Commun. 2022 Dec 06. 13(1): 7510

DAP5 enables main ORF translation on mRNAs with structured and uORF-containing 5' leaders.

Ramona Weber, Leon Kleemann, Insa Hirschberg, Min-Yi Chung, Eugene Valkov, Cátia Igreja.

Half of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here, we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is required for translation initiation on select transcripts. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated translation occurs on messenger RNAs (mRNAs) with long, structure-prone 5' leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates main CDS, but not uORF, translation suggesting a role for DAP5 in translation re-initiation. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

DOI: https://doi.org/10.1038/s41467-022-35019-5
Commun Biol. 2022 Dec 06. 5(1): 1335

The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis.

Qin Li, Yanfang Ma, Fen Chang, Yongjie Xu, Jingcheng Deng, Junyi Duan, Wei Jiang, Qihua He, Luzheng Xu, Lijun Zhong, Genze Shao, Li Li.

Faithful chromosome segregation requires bi-oriented kinetochore-microtubule attachment on the metaphase spindle. Aurora B kinase, the catalytic core of the chromosome passage complex (CPC), plays a crucial role in this process. Aurora B activation has widely been investigated in the context of protein phosphorylation. Here, we report that Aurora B is ubiquitinated in mitosis through lysine-63 ubiquitin chains (K63-Ub), which is required for its activation. Mutation of Aurora B at its primary K63 ubiquitin site inhibits its activation, reduces its kinase activity, and disrupts the association of Aurora B with other components of CPC, leading to severe mitotic defects and cell apoptosis. Moreover, we identify that BRCC36 isopeptidase complex (BRISC) is the K63-specific deubiquitinating enzyme for Aurora B. BRISC deficiency augments the accumulation of Aurora B K63-Ubs, leading to Aurora B hyperactivation and erroneous chromosome-microtubule attachments. These findings define the role of K63-linked ubiquitination in regulating Aurora B activation and provide a potential site for Aurora B-targeting drug design.

DOI: https://doi.org/10.1038/s42003-022-04299-4
Autophagy. 2022 Dec 05. 1-22

Substitution of PINK1 Gly411 modulates substrate receptivity and turnover.

Fabienne C Fiesel, Dominika Fričová, Caleb S Hayes, Mathew A Coban, Roman Hudec, Jenny M Bredenberg, Benjamin J Broadway, Briana N Markham, Tingxiang Yan, Paige K Boneski, Gabriella Fiorino, Jens O Watzlawik, Xu Hou, Arthur M McCarty, Laura J Lewis-Tuffin, Jun Zhong, Benjamin J Madden, Alban Ordureau, Heeseon An, Andreas Puschmann, Zbigniew K Wszolek, Owen A Ross, J Wade Harper, Thomas R Caulfield, Wolfdieter Springer.

  The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development.Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.

Keywords:  Mitophagy; PINK1; PRKN; parkin; parkinson disease; ubiquitin

DOI:  https://doi.org/10.1080/15548627.2022.2151294
J Biol Chem. 2022 Dec 05. pii: S0021-9258(22)01217-0. [Epub ahead of print] 102774

Arginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate.

Viacheslav Kriachkov, Angelique R Ormsby, Eric P Kusnadi, Hamish E G McWilliam, Justine D Mintern, Shanika L Amarasinghe, Matthew E Ritchie, Luc Furic, Danny M Hatters.

  Hexanucleotide expansion mutations in C9ORF72 are a frequent cause of amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here we explored the molecular features of DPR-induced stalling and examined whether known mechanisms such as ribosome quality control (RQC) regulate translation elongation on sequences that encode arginine-rich DPRs. We demonstrate that arginine-rich DPRs lead to stalling in a length-dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40×Gly-Xxx DPRs shows that stalling is most pronounced when Xxx is a charged amino acid (Arg, Lys, Glu, or Asp). Through a genome-wide knockout screen, we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, act differently in the case of arginine-rich DPRs. Indeed, these findings point to a limited scope for natural regulatory responses to resolve the arginine-rich DPR stalls, even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as a source of stress and toxicity and may be a crucial component in pathomechanisms.

Keywords:  C9ORF72 ALS; RNA-protein interaction; arginine-rich dipeptide repeats; neurodegenerative disease; protein misfolding; ribosome function; ribosome stalling; translation

DOI:  https://doi.org/10.1016/j.jbc.2022.102774
Nucleic Acids Res. 2022 Dec 08. pii: gkac1140. [Epub ahead of print]

Translation-complex profiling of fission yeast cells reveals dynamic rearrangements of scanning ribosomal subunits upon nutritional stress.

Caia Deborah Suzanne Duncan, Juan Mata.

Control of mRNA translation is key for stress responses. Translation initiation is usually rate-limiting and, in eukaryotes, involves mRNA scanning by the small ribosomal subunit. Despite its importance, many aspects of translation in vivo have not been explored fully, especially at the transcriptome-wide level. A recent method termed translation-complex profiling (TCP-seq) allows transcriptome-wide views of scanning ribosomal subunits. We applied TCP-seq to nutritional stress in the fission yeast Schizosaccharomyces pombe. At initiation sites, we observed multiple complexes resembling those of mammals, and consistent with queuing of scanning subunits. In 5' UTRs, small subunit accumulations were common and may reflect impediments to scanning. A key mediator of stress responses in S. pombe is the Fil1 transcription factor, which is regulated translationally by a poorly-understood mechanism involving upstream Open Reading Frames (uORFs). TCP-seq data of fil1 shows that stress allows scanning subunits to by-pass specific uORFs and reach the fil1 coding sequence. The integration of these observations with reporter assays revealed that fil1 translational control is mediated by a combination of scanning reinitiation-repressive and permissive uORFs, and establishes fil1 as a model for uORF-mediated translational control. Altogether, our transcriptome-wide study reveals general and gene-specific features of translation in a model eukaryote.

DOI: https://doi.org/10.1093/nar/gkac1140
Elife. 2022 Dec 07. pii: e79116. [Epub ahead of print]11

Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation.

Zhanwu Zhu, Jingjing Liu, Huan Feng, Yanning Zhang, Ruiqi Huang, Qiaochu Pan, Jing Nan, Ruidong Miao, Bo Cheng.

  Dynamic regulation of transcription is crucial for the cellular responses to various environmental or developmental cues. Gdown1 is a ubiquitously expressed, RNA polymerase II (Pol II) interacting protein, essential for the embryonic development of metazoan. It tightly binds Pol II in vitro and competitively blocks the binding of TFIIF and possibly other transcriptional regulatory factors, yet its cellular functions and regulatory circuits remain unclear. Here, we show that human GDOWN1 strictly localizes in the cytoplasm of various types of somatic cells and exhibits a potent resistance to the imposed driving force for its nuclear localization. Combined with the genetic and microscope-based approaches, two types of the functionally coupled and evolutionally conserved localization regulatory motifs are identified, including the CRM1-dependent nucleus export signal (NES) and a novel Cytoplasmic Anchoring Signal (CAS) that mediates its retention outside of the nuclear pore complexes (NPC). Mutagenesis of CAS alleviates GDOWN1's cytoplasmic retention, thus unlocks its nucleocytoplasmic shuttling properties, and the increased nuclear import and accumulation of GDOWN1 results in a drastic reduction of both Pol II and its associated global transcription levels. Importantly, the nuclear translocation of GDOWN1 occurs in response to the oxidative stresses, and the ablation of GDOWN1 significantly weakens the cellular tolerance. Collectively, our work uncovers the molecular basis of GDOWN1's subcellular localization and a novel cellular strategy of modulating global transcription and stress-adaptation via controlling the nuclear translocation of GDOWN1.

Keywords:  GDOWN1; RNA polymerase II; cell biology; chromosomes; gene expression; human; mouse; nucleocytoplasmic shuttling; stress adaptive mechanism; transcriptional regulation

DOI:  https://doi.org/10.7554/eLife.79116
Curr Protoc. 2022 Dec;2(12): e611

Targeted Protein Degradation: Design Considerations for PROTAC Development.

Nathan L Tran, Georges A Leconte, Fleur M Ferguson.

  Targeted protein degradation has recently gained widespread interest as both a novel therapeutic strategy and a useful tool in biomedical research. Targeted protein degraders are often sub-stoichiometric and do not require strong binding affinity for their targets, enabling access to previously inaccessible targets. Proteolysis-targeting chimeras (PROTACs) are one class of targeted protein degraders that promote degradation by recruiting a target protein to an E3-ligase complex via a heterobifunctional molecule. The modular nature of PROTACs allows for their rational design and systematic optimization. Here we suggest resources and methodologies for developing PROTAC degraders for researchers that may be new to the field. © 2022 Wiley Periodicals LLC.

Keywords:  PROTAC; compound optimization; medicinal chemistry; proteolysis-targeting chimera; targeted protein degradation

DOI:  https://doi.org/10.1002/cpz1.611
Cell. 2022 Dec 08. pii: S0092-8674(22)01457-X. [Epub ahead of print]185(25): 4770-4787.e20

Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT.

Daniel Gestaut, Yanyan Zhao, Junsun Park, Boxue Ma, Alexander Leitner, Miranda Collier, Grigore Pintilie, Soung-Hun Roh, Wah Chiu, Judith Frydman.

  The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

Keywords:  TRiC/CCT; XL-MS; chaperone; chaperonin; cryo-EM; prefoldin; tubulin

DOI:  https://doi.org/10.1016/j.cell.2022.11.014
Cell Rep. 2022 Dec 06. pii: S2211-1247(22)01643-6. [Epub ahead of print]41(10): 111760

Multiple sub-state structures of SERCA2b reveal conformational overlap at transition steps during the catalytic cycle.

Yuxia Zhang, Chigusa Kobayashi, Xiaohan Cai, Satoshi Watanabe, Akihisa Tsutsumi, Masahide Kikkawa, Yuji Sugita, Kenji Inaba.

  Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ into the endoplasmic reticulum (ER). Herein, we present cryo-electron microscopy (EM) structures of three intermediates of SERCA2b: Ca2+-bound phosphorylated (E1P·2Ca2+) and Ca2+-unbound dephosphorylated (E2·Pi) intermediates and another between the E2P and E2·Pi states. Our cryo-EM analysis demonstrates that the E1P·2Ca2+ state exists in low abundance and preferentially transitions to an E2P-like structure by releasing Ca2+ and that the Ca2+ release gate subsequently undergoes stepwise closure during the dephosphorylation processes. Importantly, each intermediate adopts multiple sub-state structures including those like the next one in the catalytic series, indicating conformational overlap at transition steps, as further substantiated by atomistic molecular dynamic simulations of SERCA2b in a lipid bilayer. The present findings provide insight into how enzymes accelerate catalytic cycles.

Keywords:  CP: Molecular biology; SERCA; SERCA2b; calcium; cryo-EM single-particle analysis; endoplasmic reticulum; molecular dynamics simulation

DOI:  https://doi.org/10.1016/j.celrep.2022.111760
Nat Commun. 2022 Dec 06. 13(1): 7506

Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma.

Michelle M Kameda-Smith, Helen Zhu, En-Ching Luo, Yujin Suk, Agata Xella, Brian Yee, Chirayu Chokshi, Sansi Xing, Frederick Tan, Raymond G Fox, Ashley A Adile, David Bakhshinyan, Kevin Brown, William D Gwynne, Minomi Subapanditha, Petar Miletic, Daniel Picard, Ian Burns, Jason Moffat, Kamil Paruch, Adam Fleming, Kristin Hope, John P Provias, Marc Remke, Yu Lu, Tannishtha Reya, Chitra Venugopal, Jüri Reimand, Robert J Wechsler-Reya, Gene W Yeo, Sheila K Singh.

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

DOI: https://doi.org/10.1038/s41467-022-35118-3
mBio. 2022 Dec 08. e0306822

The IRE1α-XBP1 Signaling Axis Promotes Glycolytic Reprogramming in Response to Inflammatory Stimuli.

Bevin C English, Hannah P Savage, Scott P Mahan, Vladimir E Diaz-Ochoa, Briana M Young, Basel H Abuaita, Gautam Sule, Jason S Knight, Mary X O'Riordan, Andreas J Bäumler, Renée M Tsolis.

  Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the endoplasmic reticulum (ER) stress sensor Inositol-requiring enzyme 1 alpha (IRE1α) and its downstream transcription factor X box binding protein 1 (XBP1) enhance the upregulation of glycolysis in classically activated macrophages (CAMs). The IRE1α-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by lipopolysaccharide (LPS) stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1α activation; while Toll-like receptor 4 (TLR4) supports glycolysis under both conditions, TLR4 is required for activation of IRE1α in response to LPS treatment but not B. abortus infection. Though IRE1α and XBP1 are necessary for maximal induction of glycolysis in CAMs, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cell's metabolic response and that IRE1α activation may be a way to fine-tune metabolic reprogramming. IMPORTANCE The immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1α, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1α by independent mechanisms. While the IRE1α-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1α and XBP1 in innate immunity.

Keywords:  Brucella; endoplasmic reticulum; immunometabolism; innate immunity

DOI:  https://doi.org/10.1128/mbio.03068-22
Curr Opin Chem Biol. 2022 Dec 06. pii: S1367-5931(22)00118-1. [Epub ahead of print]72 102233

Proximity labeling technologies to illuminate glycan-protein interactions.

Abigail E Reeves, Mia L Huang.

Glycosylation is a ubiquitous post-translational modification read by glycan-binding proteins (GBP) to encode important functions, but a robust understanding of these interactions and their consequences can be challenging to uncover. Glycan-GBP interactions are transient and weak, making them difficult to capture, and glycosylation is dynamic and heterogenous, necessitating study in native cellular environments to identify endogenous ligands. Proximity labeling, an experimental innovation that labels biomolecules close to a protein of interest, has recently emerged as a powerful strategy to overcome these limitations, allowing interactors to be tagged in cells for subsequent enrichment and identification by mass spectrometry-based proteomics. We will describe this nascent technique and discuss its applications in the last five years with different GBP classes, including Siglecs, galectins, and non-human lectins.

DOI: https://doi.org/10.1016/j.cbpa.2022.102233
Wiley Interdiscip Rev RNA. 2022 Dec 07. e1770

Regulation of ribonucleoprotein condensates by RNase L during viral infection.

James M Burke.

  In response to viral infection, mammalian cells activate several innate immune pathways to antagonize viral gene expression. Upon recognition of viral double-stranded RNA, protein kinase R (PKR) phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α) on serine 51. This inhibits canonical translation initiation, which broadly antagonizes viral protein synthesis. It also promotes the assembly of cytoplasmic ribonucleoprotein complexes termed stress granules (SGs). SGs are widely thought to promote cell survival and antiviral signaling. However, co-activation of the OAS/RNase L antiviral pathway inhibits the assembly of SGs and promotes the assembly of an alternative ribonucleoprotein complex termed an RNase L-dependent body (RLB). The formation of RLBs has been observed in response to double-stranded RNA, dengue virus infection, or SARS-CoV-2 infection. Herein, we review the distinct biogenesis pathways and properties of SGs and RLBs, and we provide perspective on their potential functions during the antiviral response. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Turnover and Surveillance > Regulation of RNA Stability RNA Export and Localization > RNA Localization.

Keywords:  RNase L; RNase L-dependent body; innate immunity; stress granules; virus

DOI:  https://doi.org/10.1002/wrna.1770
Cancer Res. 2022 Dec 09. pii: CAN-22-2709. [Epub ahead of print]

Protein phosphatase 1 subunit PPP1R14B stabilizes STMN1 to promote progression and paclitaxel resistance in triple-negative breast cancer.

Li Liao, Yin-Ling Zhang, Ling Deng, Chao Chen, Xiao-Yan Ma, Lisa Andriani, Shao-Ying Yang, Shu-Yuan Hu, Fang-Lin Zhang, Zhi-Ming Shao, Da-Qiang Li.

Triple-negative breast cancer (TNBC) represents the most lethal subtype of breast cancer due to its aggressive clinical features and the lack of effective therapeutic targets. To identify novel approaches for targeting TNBC, we examined the role of protein phosphatases in TNBC progression and chemoresistance. Protein phosphatase 1 regulatory subunit 14B (PPP1R14B), a poorly defined member of the protein phosphatase 1 regulatory subunits, was aberrantly upregulated in TNBC tissues and predicted poor prognosis. PPP1R14B was degraded mainly through the ubiquitin-proteasome pathway. RPS27A recruited deubiquitinase USP9X to deubiquitinate and stabilize PPP1R14B, resulting in overexpression of PPP1R14B in TNBC tissues. Gain- and loss-of-function assays demonstrated that PPP1R14B promoted TNBC cell proliferation, colony formation, migration, invasion, and resistance to paclitaxel in vitro. PPP1R14B also induced xenograft tumor growth, lung metastasis and paclitaxel resistance in vivo. Mechanistic investigations revealed that PPP1R14B maintained phosphorylation and stability of oncoprotein stathmin 1 (STMN1), a microtubule-destabilizing phosphoprotein critically involved in cancer progression and paclitaxel resistance, which was dependent on PP1 catalytic subunits α and γ. Importantly, the tumor suppressive effects of PPP1R14B deficiency could be partially rescued by ectopic expression of wild-type but not phosphorylation-deficient STMN1. Moreover, PPP1R14B increased STMN1-mediated α-tubulin acetylation, microtubule stability, and cell-cycle progression, leading to resistance of TNBC cells to paclitaxel. Collectively, these findings uncover a functional and mechanistic role of PPP1R14B in TNBC progression and paclitaxel resistance, indicating PPP1R14B is a potential therapeutic target for TNBC.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-2709
Mol Oncol. 2022 Dec 10.

Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Haris Babačić, Silvia Galardi, Husen M Umer, Mats Hellström, Lene Uhrbom, Nagaprathyusha Maturi, Deborah Cardinali, Serena Pellegatta, Alessandro Michienzi, Gianluca Trevisi, Annunziato Mangiola, Janne Lehtiö, Silvia Anna Ciafrè, Maria Pernemalm.

  Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry. We quantified >10,000 proteins in two independent GSC panels and propose a GSC-associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC-like), and another by proteins upregulated in mesenchymal GSCs (GM-like). The GM-like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non-canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non-protein-coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non-canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.

Keywords:  glioblastoma stem cells; mesenchymal; proneural; proteogenomics; proteomics; signature

DOI:  https://doi.org/10.1002/1878-0261.13355