bims-proteo 2024-06-16 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–06–16
27 papers selected by
Eric Chevet, INSERM

Tau fibrils evade autophagy by excessive p62 coating and TAX1BP1 exclusion.
FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy.
Exploring the molecular composition of the multipass translocon in its native membrane environment.
K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.
Dipeptidyl peptidases and E3 ligases of N-degron pathways cooperate to regulate protein stability.
Partitioning into ER membrane microdomains impacts autophagic protein turnover during cellular aging.
Bacterial esterases reverse lipopolysaccharide ubiquitylation to block host immunity.
Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.
UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.
The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis.
The proteostasis interactomes of trafficking-deficient variants of the voltage-gated potassium channel KV11.1 associated with Long QT Syndrome.
An autoinhibitory switch of the LSD1 disordered region controls enhancer silencing.
Liver cancer initiation requires translational activation by an oncofetal regulon involving LIN28 proteins.
Atlastin-1 regulates endosomal tubulation and lysosomal proteolysis in human cortical neurons.
Protocol for the comprehensive biochemical and cellular profiling of small-molecule degraders using CoraFluor TR-FRET technology.
Predicted mechanistic impacts of human protein missense variants.
Mechanisms of USP18 deISGylation revealed by comparative analysis with its human paralog USP41.
Bifunctional Molecules That Induce Both Targeted Degradation and Transcytosis of Extracellular Proteins in Brain Cells.
The nucleolar phase of signal recognition particle assembly.
The tidyomics ecosystem: enhancing omic data analyses.
Tamoxifen modulates nutrition deprivation-induced ER stress through AMPK-mediated ER-phagy in breast cancer cells.
Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells.
Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP.
RNATACs: Multispecific small molecules targeting RNA by induced proximity.
FoldPAthreader: predicting protein folding pathway using a novel folding force field model derived from known protein universe.
Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles.
Schlafen 11 triggers innate immune responses through its ribonuclease activity upon detection of single-stranded DNA.

Sci Adv. 2024 Jun 14. 10(24): eadm8449

Tau fibrils evade autophagy by excessive p62 coating and TAX1BP1 exclusion.

Luca Ferrari, Bernd Bauer, Yue Qiu, Martina Schuschnig, Sigrid Klotz, Dorothea Anrather, Thomas Juretschke, Petra Beli, Ellen Gelpi, Sascha Martens.

The accumulation of protein aggregates is a hallmark of many diseases, including Alzheimer's disease. As a major pillar of the proteostasis network, autophagy mediates the degradation of protein aggregates. The autophagy cargo receptor p62 recognizes ubiquitin on proteins and cooperates with TAX1BP1 to recruit the autophagy machinery. Paradoxically, protein aggregates are not degraded in various diseases despite p62 association. Here, we reconstituted the recognition by the autophagy receptors of physiological and pathological Tau forms. Monomeric Tau recruits p62 and TAX1BP1 via the sequential actions of the chaperone and ubiquitylation machineries. In contrast, Tau fibrils from Alzheimer's disease brains are recognized by p62 but fail to recruit TAX1BP1. This failure is due to the masking of fibrils ubiquitin moieties by p62. Tau fibrils are resistant to deubiquitylation, and, thus, this nonproductive interaction of p62 with the fibrils is irreversible. Our results shed light on the mechanism underlying autophagy evasion by protein aggregates and their consequent accumulation in disease.

DOI: https://doi.org/10.1126/sciadv.adm8449
bioRxiv. 2024 May 30. pii: 2024.05.28.596287. [Epub ahead of print]

FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy.

Shannon M Lacy, Rebecca J Taubitz, Nicholas D Urban, Samantha N Turowski, Eric D Smith, Adam S Helms, Daniel E Michele, Matthias C Truttmann.

Cardiomyocytes require the HSP70 chaperone BiP to maintain proteostasis in the endoplasmic reticulum (ER) following cardiac stress. The adenylyl transferase (AMPylase) FICD is increasingly recognized to regulate BiP activity through the post-translational addition of an adenosine monophosphate moiety to BiP surface residues. However, the physiological impact of FICD-mediated BiP regulation in the context of cardiovascular health is unknown. Here, we find that FICD deficiency prevents pressure overload-associated heart failure, hypertrophy, and fibrosis, and that FICD knockout mice maintain normal cardiac function after cardiac pressure overload. At a cellular level, we observe that FICD-mediated BiP AMPylation blunts the induction of the unfolded protein response (UPR ER ) and impairs BiP interaction with FAM134B, an ER-phagy receptor, thus limiting ER-phagy induction under stress. In contrast, FICD loss significantly increases BiP-dependent UPR ER induction and ER-phagy in stressed cardiomyocytes. We also uncover cell type-specific consequences of FICD activity in response to ER stress, positioning FICD as a critical proteostasis regulator in cardiac tissue. Our results highlight a novel regulatory paradigm controlling stress resilience in cardiomyocytes and offer a rationale to consider FICD as a therapeutic target to treat cardiac hypertrophy.

DOI: https://doi.org/10.1101/2024.05.28.596287
Life Sci Alliance. 2024 Aug;pii: e202302496. [Epub ahead of print]7(8):

Exploring the molecular composition of the multipass translocon in its native membrane environment.

Max Gemmer, Marten L Chaillet, Friedrich Förster.

Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.

DOI: https://doi.org/10.26508/lsa.202302496
Mol Cell. 2024 Jun 01. pii: S1097-2765(24)00441-6. [Epub ahead of print]

K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.

Harsha Garadi Suresh, Eric Bonneil, Benjamin Albert, Carine Dominique, Michael Costanzo, Carles Pons, Myra Paz David Masinas, Ermira Shuteriqi, David Shore, Anthony K Henras, Pierre Thibault, Charles Boone, Brenda J Andrews.

  Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

Keywords:  DUB-E3 ligase interplay; intranuclear quality control compartment; protein aggregation; ribosome assembly stress response; ribosomopathies; ubiquitin homeostasis; unconventional K29-linked unanchored polyubiquitin chains

DOI:  https://doi.org/10.1016/j.molcel.2024.05.018
J Cell Biol. 2024 Aug 05. pii: e202311035. [Epub ahead of print]223(8):

Dipeptidyl peptidases and E3 ligases of N-degron pathways cooperate to regulate protein stability.

Adi Shimshon, Karin Dahan, Mor Israel-Gueta, Diana Olmayev-Yaakobov, Richard T Timms, Aizat Bekturova, Yaara Makaros, Stephen J Elledge, Itay Koren.

N-degrons are short sequences located at protein N-terminus that mediate the interaction of E3 ligases (E3s) with substrates to promote their proteolysis. It is well established that N-degrons can be exposed following protease cleavage to allow recognition by E3s. However, our knowledge regarding how proteases and E3s cooperate in protein quality control mechanisms remains minimal. Using a systematic approach to monitor the protein stability of an N-terminome library, we found that proline residue at the third N-terminal position (hereafter "P+3") promotes instability. Genetic perturbations identified the dipeptidyl peptidases DPP8 and DPP9 and the primary E3s of N-degron pathways, UBR proteins, as regulators of P+3 bearing substrate turnover. Interestingly, P+3 UBR substrates are significantly enriched for secretory proteins. We found that secretory proteins relying on a signal peptide (SP) for their targeting contain a "built-in" N-degron within their SP. This degron becomes exposed by DPP8/9 upon translocation failure to the designated compartments, thus enabling clearance of mislocalized proteins by UBRs to maintain proteostasis.

DOI: https://doi.org/10.1083/jcb.202311035
Sci Rep. 2024 06 13. 14(1): 13653

Partitioning into ER membrane microdomains impacts autophagic protein turnover during cellular aging.

Simon Prokisch, Sabrina Büttner.

Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.

DOI: https://doi.org/10.1038/s41598-024-64493-8
Cell Host Microbe. 2024 Jun 12. pii: S1931-3128(24)00132-X. [Epub ahead of print]32(6): 913-924.e7

Bacterial esterases reverse lipopolysaccharide ubiquitylation to block host immunity.

Magdalena Szczesna, Yizhou Huang, Rachel E Lacoursiere, Francesca Bonini, Vito Pol, Fulya Koc, Beatrice Ward, Paul P Geurink, Jonathan N Pruneda, Teresa L M Thurston.

  Aspects of how Burkholderia escape the host's intrinsic immune response to replicate in the cell cytosol remain enigmatic. Here, we show that Burkholderia has evolved two mechanisms to block the activity of Ring finger protein 213 (RNF213)-mediated non-canonical ubiquitylation of bacterial lipopolysaccharide (LPS), thereby preventing the initiation of antibacterial autophagy. First, Burkholderia's polysaccharide capsule blocks RNF213 association with bacteria and second, the Burkholderia deubiquitylase (DUB), TssM, directly reverses the activity of RNF213 through a previously unrecognized esterase activity. Structural analysis provides insight into the molecular basis of TssM esterase activity, allowing it to be uncoupled from its isopeptidase function. Furthermore, a putative TssM homolog also displays esterase activity and removes ubiquitin from LPS, establishing this as a virulence mechanism. Of note, we also find that additional immune-evasion mechanisms exist, revealing that overcoming this arm of the host's immune response is critical to the pathogen.

Keywords:  Autophagy; Burkholderia; RNF213; TssM; Ubiquitin esterase; bacterial effector; cell-autonomous immunity; non-canonical ubiquitylation

DOI:  https://doi.org/10.1016/j.chom.2024.04.012
bioRxiv. 2024 May 31. pii: 2024.05.28.596281. [Epub ahead of print]

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.

Travis J Morgenstern, Arden Darko-Boateng, Emmanuel Afriyie, Sri Karthika Shanmugam, Xinle Zhou, Papiya Choudhury, Meera Desai, Robert S Kass, Oliver B Clarke, Henry M Colecraft.

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis- the E2 binding site, the catalytic cysteine, and the ubiquitin exosite- as revealed by a 3.1 Å cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and Ca V 2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.

DOI: https://doi.org/10.1101/2024.05.28.596281
Nat Commun. 2024 Jun 12. 15(1): 5032

UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

Zeliha Yalçin, Shiu Yeung Lam, Marieke H Peuscher, Jaco van der Torre, Sha Zhu, Prasanna V Iyengar, Daniel Salas-Lloret, Inge de Krijger, Nathalie Moatti, Ruben van der Lugt, Mattia Falcone, Aurora Cerutti, Onno B Bleijerveld, Liesbeth Hoekman, Román González-Prieto, Jacqueline J L Jacobs.

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.

DOI: https://doi.org/10.1038/s41467-024-49431-6
EMBO Rep. 2024 Jun 14.

The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis.

Helena Bragulat-Teixidor, Keisuke Ishihara, Gréta Martina Szücs, Shotaro Otsuka.

  Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.

Keywords:  Correlative Light-electron Microscopy; Endoplasmic Reticulum; Membrane Contact Site; Nuclear Envelope; Open Mitosis

DOI:  https://doi.org/10.1038/s44319-024-00175-w
J Biol Chem. 2024 Jun 12. pii: S0021-9258(24)01966-5. [Epub ahead of print] 107465

The proteostasis interactomes of trafficking-deficient variants of the voltage-gated potassium channel KV11.1 associated with Long QT Syndrome.

Christian L Egly, Lea A Barny, Tri Do, Eli F McDonald, Bjorn C Knollmann, Lars Plate.

  The voltage-gated potassium ion channel KV11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which pharmacological chaperones like E-4031 can rescue. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes of wild-type (WT) KV11.1 or trafficking-deficient channel variants in the presence or absence of E4031. We identified 572 core KV11.1 protein interactors. Trafficking-deficient variants KV11.1-G601S and KV11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We confirmed previous findings that the proteasome is critical for KV11.1 degradation. Our report provides the first comprehensive characterization of protein quality control mechanisms of KV11.1. We find extensive interactome remodeling associated with trafficking-deficient KV11.1 variants, and with pharmacological chaperone rescue of KV11.1 cell surface expression. The identified protein interactions could be targeted therapeutically to improve KV11.1 trafficking and treat Long QT Syndrome.

Keywords:  Long QT Syndrome; hERG; interactome; ion channel; mass spectrometry; trafficking

DOI:  https://doi.org/10.1016/j.jbc.2024.107465
Mol Cell. 2024 Jun 10. pii: S1097-2765(24)00440-4. [Epub ahead of print]

An autoinhibitory switch of the LSD1 disordered region controls enhancer silencing.

Amanda L Waterbury, Hui Si Kwok, Ceejay Lee, Domenic N Narducci, Allyson M Freedy, Cindy Su, Shaunak Raval, Andrew H Reiter, William Hawkins, Kwangwoon Lee, Jiaming Li, Samuel M Hoenig, Michael E Vinyard, Philip A Cole, Anders S Hansen, Steven A Carr, Malvina Papanastasiou, Brian B Liau.

  Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate.

Keywords:  autoinhibitory switch; condensate; intrinsically disordered region; leukemic differentiation; transcription factors; transcriptional corepressor

DOI:  https://doi.org/10.1016/j.molcel.2024.05.017
J Clin Invest. 2024 Jun 13. pii: e165734. [Epub ahead of print]

Liver cancer initiation requires translational activation by an oncofetal regulon involving LIN28 proteins.

Meng-Hsiung Hsieh, Yonglong Wei, Lin Li, Liem H Nguyen, Yu-Hsuan Lin, Jung M Yoon, Xuxu Sun, Xun Wang, Xin Luo, Sarah K Knutson, Christina Bracken, George Q Daley, John T Powers, Hao Zhu.

  It is unknown which post-transcriptional regulatory mechanisms are required for oncogenic competence. Here, we show that the LIN28 family of RNA-binding proteins (RBPs), which facilitate post-transcriptional RNA metabolism within ribonucleoprotein networks, are essential for the initiation of diverse oncotypes of hepatocellular carcinoma (HCC). In HCC models driven by NRASG12V/Tp53, CTNNB1/YAP/Tp53, or AKT/Tp53, mice without Lin28a and Lin28b were markedly impaired in cancer initiation. We biochemically defined an oncofetal regulon of 15 factors connected to Lin28 through direct mRNA and protein interactions. Interestingly, all were RBPs and only 1 of 15 is a Let-7 target. Polysome profiling and reporter assays showed that LIN28B directly increased the translation of 8 of these 15 RBPs. As expected, overexpression of LIN28B and IGFBP1-3 were able to genetically rescue cancer initiation. Using this platform to probe components downstream of LIN28, we found that 8 target RBPs were able to restore NRASG12V/Tp53 cancer formation in Lin28a/b deficient mice. Furthermore, these LIN28B targets promote cancer initiation through an increase in protein synthesis. LIN28B, central to an RNP regulon that increases translation of RBPs, is important for tumor initiation in the liver.

Keywords:  Hepatology; Liver cancer; Translation

DOI:  https://doi.org/10.1172/JCI165734
Neurobiol Dis. 2024 Jun 06. pii: S0969-9961(24)00155-4. [Epub ahead of print] 106556

Atlastin-1 regulates endosomal tubulation and lysosomal proteolysis in human cortical neurons.

Eliska Zlamalova, Catherine Rodger, Francesca Greco, Samuel R Cheers, Julia Kleniuk, Aishwarya G Nadadhur, Zuzana Kadlecova, Evan Reid.

  Mutation of the ATL1 gene is one of the most common causes of hereditary spastic paraplegia (HSP), a group of genetic neurodegenerative conditions characterised by distal axonal degeneration of the corticospinal tract axons. Atlastin-1, the protein encoded by ATL1, is one of three mammalian atlastins, which are homologous dynamin-like GTPases that control endoplasmic reticulum (ER) morphology by fusing tubules to form the three-way junctions that characterise ER networks. However, it is not clear whether atlastin-1 is required for correct ER morphology in human neurons and if so what the functional consequences of lack of atlastin-1 are. Using CRISPR-inhibition we generated human cortical neurons lacking atlastin-1. We demonstrate that ER morphology was altered in these neurons, with a reduced number of three-way junctions. Neurons lacking atlastin-1 had longer endosomal tubules, suggestive of defective tubule fission. This was accompanied by reduced lysosomal proteolytic capacity. As well as demonstrating that atlastin-1 is required for correct ER morphology in human neurons, our results indicate that lack of a classical ER-shaping protein such as atlastin-1 may cause altered endosomal tubulation and lysosomal proteolytic dysfunction. Furthermore, they strengthen the idea that defective lysosome function contributes to the pathogenesis of a broad group of HSPs, including those where the primary localisation of the protein involved is not at the endolysosomal system.

Keywords:  Atlastin; Endoplasmic reticulum morphology; Endosomal traffic; Endosomal tubulation; Hereditary spastic paraplegia; Lysosomal proteolysis; Lysosome

DOI:  https://doi.org/10.1016/j.nbd.2024.106556
STAR Protoc. 2024 Jun 09. pii: S2666-1667(24)00294-6. [Epub ahead of print]5(2): 103129

Protocol for the comprehensive biochemical and cellular profiling of small-molecule degraders using CoraFluor TR-FRET technology.

N Connor Payne, Saki Ichikawa, Christina M Woo, Ralph Mazitschek.

  Comprehensive characterization of small-molecule degraders, including binary and ternary complex formation and degradation efficiency, is critical for bifunctional ligand development and understanding structure-activity relationships. Here, we present a protocol for the biochemical and cellular profiling of small-molecule degraders based on CoraFluor time-resolved fluorescence resonance energy transfer (TR-FRET) technology. We describe steps for labeling antibodies and proteins, tracer saturation binding, binary target engagement, ternary complex profiling, and off-rate determination. We then detail procedures for the quantification of endogenous and GFP fusion proteins in cell lysates. For complete details on the use and execution of this protocol, please refer to Ichikawa et al.1.

Keywords:  Cell-based Assays; High Throughput Screening; Molecular/Chemical Probes

DOI:  https://doi.org/10.1016/j.xpro.2024.103129
bioRxiv. 2024 May 29. pii: 2024.05.29.596373. [Epub ahead of print]

Predicted mechanistic impacts of human protein missense variants.

Jürgen Jänes, Marc Müller, Senthil Selvaraj, Diogo Manoel, James Stephenson, Catarina Gonçalves, Aleix Lafita, Benjamin Polacco, Kirsten Obernier, Kaur Alasoo, Manuel C Lemos, Nevan Krogan, Maria Martin, Luis R Saraiva, David Burke, Pedro Beltrao.

Genome sequencing efforts have led to the discovery of tens of millions of protein missense variants found in the human population with the majority of these having no annotated role and some likely contributing to trait variation and disease. Sequence-based artificial intelligence approaches have become highly accurate at predicting variants that are detrimental to the function of proteins but they do not inform on mechanisms of disruption. Here we combined sequence and structure-based methods to perform proteome-wide prediction of deleterious variants with information on their impact on protein stability, protein-protein interactions and small-molecule binding pockets. AlphaFold2 structures were used to predict approximately 100,000 small-molecule binding pockets and stability changes for over 200 million variants. To inform on protein-protein interfaces we used AlphaFold2 to predict structures for nearly 500,000 protein complexes. We illustrate the value of mechanism-aware variant effect predictions to study the relation between protein stability and abundance and the structural properties of interfaces underlying trans protein quantitative trait loci (pQTLs). We characterised the distribution of mechanistic impacts of protein variants found in patients and experimentally studied example disease linked variants in FGFR1.

DOI: https://doi.org/10.1101/2024.05.29.596373
bioRxiv. 2024 May 28. pii: 2024.05.28.596309. [Epub ahead of print]

Mechanisms of USP18 deISGylation revealed by comparative analysis with its human paralog USP41.

Thomas Bonacci, Derek L Bolhuis, Nicholas G Brown, Michael J Emanuele.

The ubiquitin-like protein ISG15 (interferon- s timulated g ene 15 ) regulates the host response to bacterial and viral infections through its conjugation to proteins (ISGylation) following interferon production. ISGylation is antagonized by the highly specific cysteine protease USP18, which is the major deISGylating enzyme. However, mechanisms underlying USP18's extraordinary specificity towards ISG15 remains elusive. Here, we show that USP18 interacts with its paralog USP41, whose catalytic domain shares 97% identity with USP18. However, USP41 does not act as a deISGylase, which led us to perform a comparative analysis to decipher the basis for this difference, revealing molecular determinants of USP18's specificity towards ISG15. We found that USP18 C-terminus, as well as a conserved Leucine at position 198, are essential for its enzymatic activity and likely act as functional surfaces based on AlphaFold predictions. Finally, we propose that USP41 antagonizes conjugation of the understudied ubiquitin-like protein FAT10 (HLA- F a djacent transcript 10 ) from substrates in a catalytic-independent manner. Altogether, our results offer new insights into USP18's specificity towards ISG15, while identifying USP41 as a negative regulator of FAT10 conjugation.

DOI: https://doi.org/10.1101/2024.05.28.596309
J Am Chem Soc. 2024 Jun 10.

Bifunctional Molecules That Induce Both Targeted Degradation and Transcytosis of Extracellular Proteins in Brain Cells.

Rebecca A Howell, Shikun Wang, Mihir Khambete, David M McDonald, David A Spiegel.

Targeted protein degradation (TPD) has emerged as an effective therapeutic strategy for a wide range of diseases; however, the blood-brain barrier (BBB) limits access of degraders into the central nervous system (CNS). Here, we present a new class of bifunctional small molecules, called TransMoDEs (Transcytosis-inducing molecular degraders of extracellular proteins), capable of both (1) removal of target protein via lysosomal proteolysis and (2) transcytosis of protein targets across brain endothelial cells. TransMoDEs are derived from Angiopep-2, a peptide motif previously employed as a covalent tag to facilitate receptor-mediated transcytosis across the BBB. We demonstrate that TransMoDEs containing either a biotin or chloroalkane ligand can trigger endocytosis of streptavidin or HaloTag protein, respectively. Interestingly, although low-density lipoprotein receptor-related protein 1 (LRP1) has been reported as the primary receptor for Angiopep-2, TransMoDE-mediated target uptake does not rely exclusively on this pathway. Furthermore, TransMoDE-mediated endocytosis of streptavidin in a bEnd.3 BBB model occurs in a clathrin-mediated mechanism and results in both lysosomal localization and transcytosis of the target protein. This study demonstrates that TransMoDEs can recruit, transcytose, and degrade proteins of interest in cells relevant to the CNS, supporting their further development for the removal of pathogenic neuroproteins.

DOI: https://doi.org/10.1021/jacs.3c13320
Life Sci Alliance. 2024 Aug;pii: e202402614. [Epub ahead of print]7(8):

The nucleolar phase of signal recognition particle assembly.

Amani Issa, Florence Schlotter, Justine Flayac, Jing Chen, Ludivine Wacheul, Manon Philippe, Lucas Sardini, Lalia Mostefa, Franck Vandermoere, Edouard Bertrand, Céline Verheggen, Denis Lj Lafontaine, Séverine Massenet.

The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.

DOI: https://doi.org/10.26508/lsa.202402614
Nat Methods. 2024 Jun 14.

The tidyomics ecosystem: enhancing omic data analyses.

tidyomics Consortium

The growth of omic data presents evolving challenges in data manipulation, analysis and integration. Addressing these challenges, Bioconductor provides an extensive community-driven biological data analysis platform. Meanwhile, tidy R programming offers a revolutionary data organization and manipulation standard. Here we present the tidyomics software ecosystem, bridging Bioconductor to the tidy R paradigm. This ecosystem aims to streamline omic analysis, ease learning and encourage cross-disciplinary collaborations. We demonstrate the effectiveness of tidyomics by analyzing 7.5 million peripheral blood mononuclear cells from the Human Cell Atlas, spanning six data frameworks and ten analysis tools.

DOI: https://doi.org/10.1038/s41592-024-02299-2
Breast Cancer Res Treat. 2024 Jun 14.

Tamoxifen modulates nutrition deprivation-induced ER stress through AMPK-mediated ER-phagy in breast cancer cells.

Biswas Bidisha, Manickavasagan Sowmya, Suchita Shalini, Chandrasekaran Mythri, Anshu Gupta, Gangipangi Vijayakumar, Selvaraju Sudhagar.

   PURPOSE: Rapid proliferation and nutrition starvation in the tumor microenvironment pose significant challenges to cellular protein homeostasis. The accumulation of misfolded proteins in the endoplasmic reticulum lumen induces stress on cells and causes irreversible damage to cells if unresolved. Emerging reports emphasize the influence of the tumor microenvironment on therapeutic molecule efficacy and treatment outcomes. Hence, we aimed to understand the influence of tamoxifen on the cellular adaptation to endoplasmic reticulum stress during metabolic stress in breast cancer cells.
METHODS: Nutrition deprivation induces endoplasmic reticulum stress (ER stress), and the unfolded protein response (UPR) in breast cancer cells was confirmed by a Thioflavin B assay and western blotting. Tamoxifen-indued ER-phagy was studied using an MCD assay, confocal microscopy, and western blotting.
RESULTS: Nutrition deprivation induces ER stress in breast cancer cells. Interestingly, tamoxifen modulates the nutrition deprivation-induced endoplasmic reticulum stress through enhancing the selective ER-phagy, a specialized autophagy. The tamoxifen-induced ER-phagy is mediated by AMPK activation. The pharmacological inhibition of AMPK blocks tamoxifen-induced ER-phagy and tamoxifen modulatory effect on ER stress during nutrition deprivation.
CONCLUSION: Tamoxifen modulates ER stress by inducing ER-phagy through AMPK, thereby, may support breast cancer cell survival during nutrition deprivation conditions.

Keywords:  AMPK; Breast cancer; ER stress; ER-phagy; Nutrition deprivation; Tamoxifen

DOI:  https://doi.org/10.1007/s10549-024-07398-4
STAR Protoc. 2024 Jun 07. pii: S2666-1667(24)00286-7. [Epub ahead of print]5(2): 103121

Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells.

Clément Maghe, Julie Gavard.

  Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1.

Keywords:  Cancer; Cell Biology; Cell culture; Cell separation/fractionation; Molecular/Chemical Probes; Protein Biochemistry; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2024.103121
Mol Cell. 2024 Jun 05. pii: S1097-2765(24)00442-8. [Epub ahead of print]

Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP.

Yoann Nicolas, Hélène Bret, Elda Cannavo, Ananya Acharya, Petr Cejka, Valérie Borde, Raphaël Guerois.

  In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.

Keywords:  AlphaFold; DNA repair; coevolution; conformational change; double-strand breaks; homologous recombination; meiosis; nuclease; protein complex; structural models

DOI:  https://doi.org/10.1016/j.molcel.2024.05.019
Cell Chem Biol. 2024 Jun 10. pii: S2451-9456(24)00207-1. [Epub ahead of print]

RNATACs: Multispecific small molecules targeting RNA by induced proximity.

Yan Song, Jia Cui, Jiaqiang Zhu, Boseon Kim, Mei-Ling Kuo, Patrick Ryan Potts.

RNA-targeting small molecules (rSMs) have become an attractive modality to tackle traditionally undruggable proteins and expand the druggable space. Among many innovative concepts, RNA-targeting chimeras (RNATACs) represent a new class of multispecific, induced proximity small molecules that act by chemically bringing RNA targets into proximity with an endogenous RNA effector, such as a ribonuclease (RNase). Depending on the RNA effector, RNATACs can alter the stability, localization, translation, or splicing of the target RNA. Although still in its infancy, this new modality has the potential for broad applications in the future to treat diseases with high unmet need. In this review, we discuss potential advantages of RNATACs, recent progress in the field, and challenges to this cutting-edge technology.

DOI: https://doi.org/10.1016/j.chembiol.2024.05.006
Genome Biol. 2024 Jun 11. 25(1): 152

FoldPAthreader: predicting protein folding pathway using a novel folding force field model derived from known protein universe.

Kailong Zhao, Pengxin Zhao, Suhui Wang, Yuhao Xia, Guijun Zhang.

  Protein folding has become a tractable problem with the significant advances in deep learning-driven protein structure prediction. Here we propose FoldPAthreader, a protein folding pathway prediction method that uses a novel folding force field model by exploring the intrinsic relationship between protein evolution and folding from the known protein universe. Further, the folding force field is used to guide Monte Carlo conformational sampling, driving the protein chain fold into its native state by exploring potential intermediates. On 30 example targets, FoldPAthreader successfully predicts 70% of the proteins whose folding pathway is consistent with biological experimental data.

Keywords:  Evolutionary history; Folding force field; Protein folding pathway

DOI:  https://doi.org/10.1186/s13059-024-03291-x
Proc Natl Acad Sci U S A. 2024 Jun 18. 121(25): e2322588121

Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles.

Chieh-Hsiang Tan, Ting-Yu Wang, Heenam Park, Brett Lomenick, Tsui-Fen Chou, Paul W Sternberg.

  The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.

Keywords:  lysosome-related organelle; microproteomics; tissue-specific mass-spectrometry; yolk protein

DOI:  https://doi.org/10.1073/pnas.2322588121
Sci Immunol. 2024 Jun 14. 9(96): eadj5465

Schlafen 11 triggers innate immune responses through its ribonuclease activity upon detection of single-stranded DNA.

Peng Zhang, Xiaoqing Hu, Zekun Li, Qian Liu, Lele Liu, Yingying Jin, Sizhe Liu, Xiang Zhao, Jianqiao Wang, Delong Hao, Houzao Chen, Depei Liu.

Nucleic acids are major structures detected by the innate immune system. Although intracellular single-stranded DNA (ssDNA) accumulates during pathogen infection or disease, it remains unclear whether and how intracellular ssDNA stimulates the innate immune system. Here, we report that intracellular ssDNA triggers cytokine expression and cell death in a CGT motif-dependent manner. We identified Schlafen 11 (SLFN11) as an ssDNA-activated RNase, which is essential for the innate immune responses induced by intracellular ssDNA and adeno-associated virus infection. We found that SLFN11 directly binds ssDNA containing CGT motifs through its carboxyl-terminal domain, translocates to the cytoplasm upon ssDNA recognition, and triggers innate immune responses through its amino-terminal ribonuclease activity that cleaves transfer RNA (tRNA). Mice deficient in Slfn9, a mouse homolog of SLFN11, exhibited resistance to CGT ssDNA-induced inflammation, acute hepatitis, and septic shock. This study identifies CGT ssDNA and SLFN11/9 as a class of immunostimulatory nucleic acids and pattern recognition receptors, respectively, and conceptually couples DNA immune sensing to controlled RNase activation and tRNA cleavage.

DOI: https://doi.org/10.1126/sciimmunol.adj5465