bims-proteo 2023-08-06 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–08–06
thirty-six papers selected by
Eric Chevet, INSERM

Mechanisms of substrate processing during ER-associated protein degradation.
RNF26 binds perinuclear vimentin filaments to integrate ER and endolysosomal responses to proteotoxic stress.
Divergent Proteome Reactivity Influences Arm-Selective Activation of the Unfolded Protein Response by Pharmacological Endoplasmic Reticulum Proteostasis Regulators.
In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway.
To be (in a transcriptional complex) or not to be (promoting UBR5 ubiquitylation): That is an answer to how degradation controls gene expression.
Muscle specific ER-associated degradation maintains postnatal muscle hypertrophy and systemic energy metabolism.
Zinc finger 1 of the RING E3 ligase, RNF125, interacts with the E2 to enhance ubiquitylation.
The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ.
SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.
Building yeast libraries to dissect terminal degrons with fluorescent timers.
Early proteostasis of caveolins synchronizes trafficking, degradation, and oligomerization to prevent toxic aggregation.
You can "tail" them apart: paralog-specific functions of CRL4B ubiquitin ligases during mitosis and brain development.
Identification of N-degrons and N-recognins using peptide pull-downs combined with quantitative mass spectrometry.
Small-Molecule Hydrophobic Tagging: A Promising Strategy of Druglike Technology for Targeted Protein Degradation.
Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins.
O-GlcNAc Modification Alters the Chaperone Activity of HSP27 Charcot-Marie-Tooth Type 2 (CMT2) Variants in a Mutation-Selective Fashion.
LIRcentral: a manually curated online database of experimentally validated functional LIR motifs.
N-degron-mediated degradation of the proteolytically activated form of PKC-theta kinase attenuates its pro-apoptotic function.
Structural and functional complexity of HSP90 in cellular homeostasis and disease.
The Sequential Recruitments of Rab-GTPase Ypt1p and the NNS Complex onto pre-HAC1 mRNA Promote Its Nuclear Degradation in Baker's Yeast.
Analyzing the topology of N-linked glycans by PNGase F accessibility assay.
FBXO7/ntc and USP30 antagonistically set the ubiquitination threshold for basal mitophagy and provide a target for Pink1 phosphorylation in vivo.
Escherichia coli small heat shock protein IbpA plays a role in regulating the heat shock response by controlling the translation of σ32.
Mapping the MOB proteins' proximity network reveals a unique interaction between human MOB3C and the RNase P complex.
Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.
Functional analogs of mammalian 4E-BPs reveal a role for TOR in global plant translation.
Proximity-Based Modalities for Biology and Medicine.
ER stress decreases exosome production through adiponectin/T-cadherin-dependent and -independent pathways.
Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.
AI-based AlphaFold2 significantly expands the structural space of the autophagy pathway.
BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling.
Loss of PERK function promotes ferroptosis by downregulating SLC7A11 (System Xc⁻) in colorectal cancer.
Structural basis for membrane-proximal proteolysis of substrates by ADAM10.
cGAS-STING drives ageing-related inflammation and neurodegeneration.
Engineering tRNA abundances for synthetic cellular systems.
Senescent cells form nuclear foci that contain the 26S proteasome.

Nat Rev Mol Cell Biol. 2023 Aug 01.

Mechanisms of substrate processing during ER-associated protein degradation.

John C Christianson, Ernst Jarosch, Thomas Sommer.

Maintaining proteome integrity is essential for long-term viability of all organisms and is overseen by intrinsic quality control mechanisms. The secretory pathway of eukaryotes poses a challenge for such quality assurance as proteins destined for secretion enter the endoplasmic reticulum (ER) and become spatially segregated from the cytosolic machinery responsible for disposal of aberrant (misfolded or otherwise damaged) or superfluous polypeptides. The elegant solution provided by evolution is ER-membrane-bound ubiquitylation machinery that recognizes misfolded or surplus proteins or by-products of protein biosynthesis in the ER and delivers them to 26S proteasomes for degradation. ER-associated protein degradation (ERAD) collectively describes this specialized arm of protein quality control via the ubiquitin-proteasome system. But, instead of providing a single strategy to remove defective or unwanted proteins, ERAD represents a collection of independent processes that exhibit distinct yet overlapping selectivity for a wide range of substrates. Not surprisingly, ER-membrane-embedded ubiquitin ligases (ER-E3s) act as central hubs for each of these separate ERAD disposal routes. In these processes, ER-E3s cooperate with a plethora of specialized factors, coordinating recognition, transport and ubiquitylation of undesirable secretory, membrane and cytoplasmic proteins. In this Review, we focus on substrate processing during ERAD, highlighting common threads as well as differences between the many routes via ERAD.

DOI: https://doi.org/10.1038/s41580-023-00633-8
EMBO J. 2023 Jul 31. e111252

RNF26 binds perinuclear vimentin filaments to integrate ER and endolysosomal responses to proteotoxic stress.

Tom Cremer, Lenard M Voortman, Erik Bos, Marlieke Lm Jongsma, Laurens R Ter Haar, Jimmy Jll Akkermans, Cami Mp Talavera Ormeño, Ruud Hm Wijdeven, Jelle de Vries, Robbert Q Kim, George Mc Janssen, Peter A van Veelen, Roman I Koning, Jacques Neefjes, Ilana Berlin.

  Proteotoxic stress causes profound endoplasmic reticulum (ER) membrane remodeling into a perinuclear quality control compartment (ERQC) for the degradation of misfolded proteins. Subsequent return to homeostasis involves clearance of the ERQC by endolysosomes. However, the factors that control perinuclear ER integrity and dynamics remain unclear. Here, we identify vimentin intermediate filaments as perinuclear anchors for the ER and endolysosomes. We show that perinuclear vimentin filaments engage the ER-embedded RING finger protein 26 (RNF26) at the C-terminus of its RING domain. This restricts RNF26 to perinuclear ER subdomains and enables the corresponding spatial retention of endolysosomes through RNF26-mediated membrane contact sites (MCS). We find that both RNF26 and vimentin are required for the perinuclear coalescence of the ERQC and its juxtaposition with proteolytic compartments, which facilitates efficient recovery from ER stress via the Sec62-mediated ER-phagy pathway. Collectively, our findings reveal a scaffolding mechanism that underpins the spatiotemporal integration of organelles during cellular proteostasis.

Keywords:  ER stress; ERphagy; RNF26; endolysosomes; intermediate filaments

DOI:  https://doi.org/10.15252/embj.2022111252
ACS Chem Biol. 2023 Jul 31.

Divergent Proteome Reactivity Influences Arm-Selective Activation of the Unfolded Protein Response by Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Gabriel M Kline, Ryan J Paxman, Chung-Yon Lin, Nicole Madrazo, Leonard Yoon, Julia M D Grandjean, Kyunga Lee, Karina Nugroho, Evan T Powers, R Luke Wiseman, Jeffery W Kelly.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the unfolded protein response (UPR) has proven useful for ameliorating proteostasis deficiencies in cellular and mouse models of numerous etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of endoplasmic reticulum (ER) protein disulfide isomerases (PDIs). Another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent modification of proteins including multiple PDIs. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. However, the extent of PDI labeling by AA147 approaches a plateau more rapidly than PDI labeling by AA132. These observations together suggest that AA132 can access a larger pool of proteins for covalent modification, possibly because its activated form is less susceptible to quenching than activated AA147. In other words, the lower reactivity of activated AA132 allows it to persist longer and modify more PDIs in the cellular environment. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and enables selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

DOI: https://doi.org/10.1021/acschembio.3c00042
Methods Enzymol. 2023 ;pii: S0076-6879(23)00081-2. [Epub ahead of print]686 205-220

In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway.

Alexander Sandmann, Nico Dissmeyer.

  As a part of the ubiquitin-proteasome system, E3 ubiquitin ligases play an important role in the regulation of the proteome in eukaryotic cells. These enzymes are extensively studied because of their crucial function, however it can be challenging to observe E3 ubiquitin ligases in action. Here, we outline a method for determining whether a known or potential E3 ubiquitin ligase exhibits autoubiquitination activity in vitro using PROTEOLYSIS1 (PRT1, AT3G24800), the first identified N-degron pathway E3 ubiquitin ligase from plants as an example. The approach provided here makes it possible to analyze mutations that could reduce or eliminate activity, to test for interaction with E2 ubiquitin conjugating enzymes, as well as to check for in vitro substrate ubiquitination.

Keywords:  E3 ubiquitin ligase; N-degron pathway; Protein degradation; Ubiquitin

DOI:  https://doi.org/10.1016/bs.mie.2023.02.014
Mol Cell. 2023 Aug 03. pii: S1097-2765(23)00524-5. [Epub ahead of print]83(15): 2616-2618

To be (in a transcriptional complex) or not to be (promoting UBR5 ubiquitylation): That is an answer to how degradation controls gene expression.

Laura A Hehl, Brenda A Schulman.

Tsai et al.1 in this issue and Mark et al.2 in Cell reveal how the E3 ligase UBR5 mediates broad regulation by selectively targeting agonist-bound nuclear hormone receptors, MYC, and other transcriptional regulators not incorporated into active gene expression complexes.

DOI: https://doi.org/10.1016/j.molcel.2023.07.010
JCI Insight. 2023 Aug 03. pii: e170387. [Epub ahead of print]

Muscle specific ER-associated degradation maintains postnatal muscle hypertrophy and systemic energy metabolism.

Benedict Abdon, Yusheng Liang, Débora da Luz Scheffer, Mauricio Torres, Neha Shrestha, Rachel B Reinert, You Lu, Brent Pederson, Amara Bugarin-Lapuz, Sander Kersten, Ling Qi.

  The growth of skeletal muscle relies on a delicate equilibrium between protein synthesis and degradation; however, how proteostasis is managed in the endoplasmic reticulum is largely unknown. Here, we report that the SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD) complex, the primary molecular machinery that degrades misfolded proteins in the ER is vital to maintain postnatal muscle growth and systemic energy balance. Myocyte-specific SEL1L deletion blunts the hypertrophic phase of muscle growth, resulting in a net zero gain of muscle mass during this developmental period and a 30% reduction in overall body growth. In addition, myocyte-specific SEL1L deletion triggered a systemic reprogramming of metabolism characterized by improved glucose sensitivity, enhanced beiging of adipocytes, and resistance to diet induced obesity. These effects were partially mediated by the upregulation of the myokine FGF21. These findings highlight the pivotal role of SEL1L-HRD1 ERAD activity in skeletal myocytes for postnatal muscle growth, and its physiological integration in maintaining whole-body energy balance.

Keywords:  Cell Biology; Cell stress; Muscle Biology; Protein misfolding; Skeletal muscle

DOI:  https://doi.org/10.1172/jci.insight.170387
Structure. 2023 Jul 26. pii: S0969-2126(23)00249-6. [Epub ahead of print]

Zinc finger 1 of the RING E3 ligase, RNF125, interacts with the E2 to enhance ubiquitylation.

Adam J Middleton, Fareeda M Barzak, Thornton J Fokkens, Khanh Nguyen, Catherine L Day.

  Inflammation is essential for healthy immune function, wound healing, and resolution of infection. RIG-I is a key RNA sensor that initiates an immune response, with activation and termination of RIG-I signaling reliant on its modification with ubiquitin. The RING E3 ubiquitin ligase, RNF125, has a critical role in the attenuation of RIG-I signaling, yet it is not known how RNF125 promotes ubiquitin transfer or how its activity is regulated. Here we show that the E3 ligase activity of RNF125 relies on the first zinc finger (ZF1) as well as the RING domain. Surprisingly, ZF1 helps recruit the E2, while residues N-terminal to the RING domain appear to activate the E2∼Ub conjugate. These discoveries help explain how RNF125 brings about the termination of RIG-I dependent inflammatory responses, and help account for the contribution of RNF125 to disease. This study also reveals a new role for ZF domains in E3 ligases.

Keywords:  E2 activation; E3 ligase; RIG-I; RING; RNF125; degradation; ubiquitin conjugating

DOI:  https://doi.org/10.1016/j.str.2023.07.007
J Cell Biol. 2023 10 02. pii: e202301112. [Epub ahead of print]222(10):

The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ.

Lukas A Fuentes, Zach Marin, Jonathan Tyson, David Baddeley, Joerg Bewersdorf.

The endoplasmic reticulum's (ER's) structure is directly linked to the many functions of the ER, but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects the local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule, increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4.

DOI: https://doi.org/10.1083/jcb.202301112
Sci Adv. 2023 Aug 02. 9(31): eadh2073

SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.

Daniel Salas-Lloret, Nicolette S Jansen, Easa Nagamalleswari, Coen van der Meulen, Ekaterina Gracheva, Arnoud H de Ru, H Anne Marie Otte, Peter A van Veelen, Andrea Pichler, Joachim Goedhart, Alfred C O Vertegaal, Román González-Prieto.

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2, and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 6500 SUMO2/3 target proteins. The limited number of SUMO E3s provides the unique opportunity to systematically study E3 substrate wiring. We developed SUMO-activated target traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL (ZNF451-3), and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool to browse the dataset in an interactive manner. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

DOI: https://doi.org/10.1126/sciadv.adh2073
Methods Enzymol. 2023 ;pii: S0076-6879(23)00079-4. [Epub ahead of print]686 297-319

Building yeast libraries to dissect terminal degrons with fluorescent timers.

Ka-Yiu Edwin Kong, Christian Reinbold, Michael Knop, Anton Khmelinskii.

  Selective degradation of unnecessary or abnormal proteins by the ubiquitin-proteasome system is an essential part of proteostasis. Ubiquitin ligases recognize substrates of selective protein degradation and modify them with polyubiquitin chains, which mark them for proteasomal degradation. Substrate recognition by ubiquitin ligases often involves degradation signals or degrons, which are typically short linear motifs found in intrinsically disordered regions, e.g., at protein termini. However, specificity in selective protein degradation is generally not well understood, as for most ubiquitin ligases no degrons have been identified thus far. To address this limitation, high-throughput mutagenesis approaches, such as multiplexed protein stability (MPS) profiling, have been developed, enabling systematic surveys of degrons in vivo or allowing to define degron motifs recognized by different ubiquitin ligases. In MPS profiling, thousands of short peptides can be assessed in parallel for their ability to trigger degradation of a fluorescent timer reporter. Here, we describe common types of libraries used to identify and dissect degrons located at protein termini using MPS profiling in budding yeast, and provide protocols for their construction.

Keywords:  C-degron; Deep mutational scanning; Degradation signal; Degron; Fluorescent timer; MPS profiling; N-degron; Terminal degron

DOI:  https://doi.org/10.1016/bs.mie.2023.02.012
J Cell Biol. 2023 09 04. pii: e202204020. [Epub ahead of print]222(9):

Early proteostasis of caveolins synchronizes trafficking, degradation, and oligomerization to prevent toxic aggregation.

Frederic Morales-Paytuví, Alba Fajardo, Carles Ruiz-Mirapeix, James Rae, Francesc Tebar, Marta Bosch, Carlos Enrich, Brett M Collins, Robert G Parton, Albert Pol.

Caveolin-1 (CAV1) and CAV3 are membrane-sculpting proteins driving the formation of the plasma membrane (PM) caveolae. Within the PM mosaic environment, caveola assembly is unique as it requires progressive oligomerization of newly synthesized caveolins while trafficking through the biosynthetic-secretory pathway. Here, we have investigated these early events by combining structural, biochemical, and microscopy studies. We uncover striking trafficking differences between caveolins, with CAV1 rapidly exported to the Golgi and PM while CAV3 is initially retained in the endoplasmic reticulum and laterally moves into lipid droplets. The levels of caveolins in the endoplasmic reticulum are controlled by proteasomal degradation, and only monomeric/low oligomeric caveolins are exported into the cis-Golgi with higher-order oligomers assembling beyond this compartment. When any of those early proteostatic mechanisms are compromised, chemically or genetically, caveolins tend to accumulate along the secretory pathway forming non-functional aggregates, causing organelle damage and triggering cellular stress. Accordingly, we propose a model in which disrupted proteostasis of newly synthesized caveolins contributes to pathogenesis.

DOI: https://doi.org/10.1083/jcb.202204020
EMBO J. 2023 Aug 02. e114931

You can "tail" them apart: paralog-specific functions of CRL4B ubiquitin ligases during mitosis and brain development.

Aparna Baxi, Achim Werner.

The CUL4 paralogs CUL4A and CUL4B assemble into structurally similar multisubunit ubiquitin E3 ligases (CRL4A/B) that regulate diverse aspects of cell biology. New work in this issue of The EMBO Journal shows that the longer N-terminal tail of CUL4B tells these molecular twins apart, by promoting the formation of paralog-specific CRL4B complexes that control cytoskeletal processes during mitosis and brain development.

DOI: https://doi.org/10.15252/embj.2023114931
Methods Enzymol. 2023 ;pii: S0076-6879(23)00063-0. [Epub ahead of print]686 67-97

Identification of N-degrons and N-recognins using peptide pull-downs combined with quantitative mass spectrometry.

Franziska Müller, Tanja Bange.

  Regulated protein degradation controls protein levels of all short-lived proteins to ensure cellular homeostasis and also protects cells from misfolded or other abnormal proteins. The most important players in the degradation system are E3 ubiquitin ligases which recognize exposed sequence motifs, so-called degrons, of target proteins and mark them through the attachment of ubiquitin for degradation. N-terminal (Nt) sequences are extensively used as degrons (N-degrons) and all 20 amino acids are able to feed proteins in 1 of the 5 known N-degron pathways. Studies have mainly focused on characterizing systematically the role of the starting amino acid on protein stability and less on the identification of the E3 ligases involved. Recent data from our lab and literature suggest that there is an extensive interplay of N-recognins and Nt-modifying enzymes like Nt-acetyltransferases (NATs) or N-myristoyltransferases which only starts to be elucidated. It suggests that improperly modified or unexpectedly unmodified proteins become rapidly removed after synthesis ensuring protein maturation and quality control of specific subsets of proteins. Here, we describe a peptide pull-down and down-stream bioinformatics workflow conducted in the MaxQuant and Perseus computational environment to identify N-recognin candidates in an unbiased way using quantitative mass spectrometry (MS)-based proteomics. Our workflow allows the identification of N-recognin candidates for specific N-degrons, to determine their sequence specificity and it can be applied as well more general to identify binding partners of N-terminal modifications. This method paves the way to identify pathways involved in protein quality control and stability acting at the N-terminus.

Keywords:  E3 ligase; Label-free quantification; Mass-spectrometry; N-degron; N-recognin; N-terminal acetylation; N-terminal modification; Peptide pull-down; Protein quality control and stability; Quantitative MS-based proteomics

DOI:  https://doi.org/10.1016/bs.mie.2023.02.007
J Med Chem. 2023 Aug 03.

Small-Molecule Hydrophobic Tagging: A Promising Strategy of Druglike Technology for Targeted Protein Degradation.

Shaowen Xie, Jingjie Zhu, Junda Li, Feiyan Zhan, Hong Yao, Jinyi Xu, Shengtao Xu.

Targeted protein degradation (TPD) technologies have catalyzed a paradigm shift in therapeutic strategies and offer innovative avenues for drug design. Hydrophobic tags (HyTs) are bifunctional TPD molecules consisting of a ″lipophilic small-molecule tags″ group and a small-molecule ligand for the target protein. Despite the vast potential of HyTs, they have received relatively limited attention as a promising frontier. Leveraging their lower molecular weight and reduced numbers of hydrogen bond donors/acceptors (HBDs/HBAs) in comparison with proteolysis-targeting chimeras (PROTACs), HyTs present a compelling approach for enhancing druglike properties. In this Perspective, we explore the diverse range of HyT structures and their corresponding degradation mechanisms, thereby illuminating their broad applicability in targeting a diverse array of proteins, including previously elusive targets. Moreover, we scrutinize the challenges and opportunities entailed in developing this technology as a viable and fruitful strategy for drug discovery.

DOI: https://doi.org/10.1021/acs.jmedchem.3c00736
Proc Natl Acad Sci U S A. 2023 Aug 08. 120(32): e2218217120

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins.

Duccio Malinverni, Stefano Zamuner, Mathieu E Rebeaud, Alessandro Barducci, Nadinath B Nillegoda, Paolo De Los Rios.

  The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

Keywords:  Hsp40 co-chaperones; J-domain proteins; artificial neural networks; large-scale data analysis; protein homeostasis

DOI:  https://doi.org/10.1073/pnas.2218217120
ACS Chem Biol. 2023 Aug 04.

O-GlcNAc Modification Alters the Chaperone Activity of HSP27 Charcot-Marie-Tooth Type 2 (CMT2) Variants in a Mutation-Selective Fashion.

Stuart P Moon, Binyou Wang, Benjamin S Ahn, Andrew H Ryu, Eldon R Hard, Afraah Javed, Matthew R Pratt.

Increased O-GlcNAc is a common feature of cellular stress, and the upregulation of this dynamic modification is associated with improved survival under these conditions. Likewise, the heat shock proteins are also increased under stress and prevent protein misfolding and aggregation. We previously linked these two phenomena by demonstrating that O-GlcNAc directly increases the chaperone of certain small heat shock proteins, including HSP27. Here, we examine this linkage further by exploring the potential function of O-GlcNAc on mutants of HSP27 that cause a heritable neuropathy called Charcot-Marie-Tooth type 2 (CMT2) disease. Using synthetic protein chemistry, we prepared five of these mutants bearing an O-GlcNAc at the major site of modification. Upon subsequent biochemical analysis of these proteins, we found that O-GlcNAc has different effects, depending on the location of the individual mutants. We believe that this has important implications for O-GlcNAc and other PTMs in the context of polymorphisms or diseases with high levels of protein mutation.

DOI: https://doi.org/10.1021/acschembio.3c00292
Autophagy. 2023 Aug 02. 1-12

LIRcentral: a manually curated online database of experimentally validated functional LIR motifs.

Agathangelos Chatzichristofi, Vasileios Sagris, Aristos Pallaris, Marios Eftychiou, Ioanna Kalvari, Nicholas Price, Theodosios Theodosiou, Ioannis Iliopoulos, Ioannis P Nezis, Vasilis J Promponas.

  Several selective macroautophagy receptor and adaptor proteins bind members of the Atg8 (autophagy related 8) family using short linear motifs (SLiMs), most often referred to as Atg8-family interacting motifs (AIMs) or LC3-interacting regions (LIRs). AIM/LIR motifs have been extensively studied during the last fifteen years, since they can uncover the underlying biological mechanisms and possible substrates for this key catabolic process of eukaryotic cells. Prompted by the fact that experimental information regarding LIR motifs can be found scattered across heterogeneous literature resources, we have developed LIRcentral (https://lircentral.eu), a freely available online repository for user-friendly access to comprehensive, high-quality information regarding LIR motifs from manually curated publications. Herein, we describe the development of LIRcentral and showcase currently available data and features, along with our plans for the expansion of this resource. Information incorporated in LIRcentral is useful for accomplishing a variety of research tasks, including: (i) guiding wet biology researchers for the characterization of novel instances of LIR motifs, (ii) giving bioinformaticians/computational biologists access to high-quality LIR motifs for building novel prediction methods for LIR motifs and LIR containing proteins (LIRCPs) and (iii) performing analyses to better understand the biological importance/features of functional LIR motifs. We welcome feedback on the LIRcentral content and functionality by all interested researchers and anticipate this work to spearhead a community effort for sustaining this resource which will further promote progress in studying LIR motifs/LIRCPs.Abbreviations: AIM, Atg8-family interacting motif; Atg8, autophagy related 8; GABARAP, GABA type A receptor-associated protein; LIR, LC3-interacting region; LIRCP, LIR-containing protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PMID, PubMed identifier; PPI, protein-protein interaction; SLiM, short linear motif.

Keywords:  Atg8 family; database; manual literature curation/annotation; online resource; peptide-protein interaction; selective macroautophagy

DOI:  https://doi.org/10.1080/15548627.2023.2235851
Cell Signal. 2023 Jul 27. pii: S0898-6568(23)00244-9. [Epub ahead of print] 110830

N-degron-mediated degradation of the proteolytically activated form of PKC-theta kinase attenuates its pro-apoptotic function.

Mohamed A Eldeeb, Wenbin Zhou, Mansoore Esmaili, Alaa M Elgohary, Hai Wei, Richard P Fahlman.

  Cellular signalling cues lead to the initiation of apoptotic pathways and often result in the activation of caspases which in turn cause the generation of proteolytically generated protein fragments with new or altered functions. Mounting number of studies reveal that the activity of these proteolytically activated protein fragments can be counteracted via their selective degradation by the N-degron degradation pathways. Here, we investigate the proteolytically generated fragment of the PKC theta kinase, where we study the first report on the stability of this pro-apoptotic protein fragment. We have determined that the pro-apoptotic cleaved fragment of PKC-theta is unstable in cells because its N-terminal lysine targets it for proteasomal degradation via the N-end rule pathway and this degradation is inhibited by mutating the destabilizing N-termini, knockdown of the UBR1 and UBR2 E3 ligases. Tellingly, we demonstrate that the metabolic stabilization of the cleaved fragment of PKC-theta or inhibition of the N-end rule augments the apoptosis-inducing effect of staurosporine in Jurkat cells. Notably, we have demonstrated that the cleaved fragment of PKC theta, per se, can induce apoptotic cell death in Jurkat T-cell leukemia. Our results expand the functional scope of mammalian N-degron pathway, and support the notion that targeting N-degron degradation machinery may have promising therapeutic implications in cancer cells.

Keywords:  Cell death; Cellular signalling; Proteasome; Protein degradation; Protein quality control; Proteolysis; Ubiquitin

DOI:  https://doi.org/10.1016/j.cellsig.2023.110830
Nat Rev Mol Cell Biol. 2023 Jul 31.

Structural and functional complexity of HSP90 in cellular homeostasis and disease.

Gabriela Chiosis, Chander S Digwal, Jane B Trepel, Len Neckers.

Heat shock protein 90 (HSP90) is a chaperone with vital roles in regulating proteostasis, long recognized for its function in protein folding and maturation. A view is emerging that identifies HSP90 not as one protein that is structurally and functionally homogeneous but, rather, as a protein that is shaped by its environment. In this Review, we discuss evidence of multiple structural forms of HSP90 in health and disease, including homo-oligomers and hetero-oligomers, also termed epichaperomes, and examine the impact of stress, post-translational modifications and co-chaperones on their formation. We describe how these variations influence context-dependent functions of HSP90 as well as its interaction with other chaperones, co-chaperones and proteins, and how this structural complexity of HSP90 impacts and is impacted by its interaction with small molecule modulators. We close by discussing recent developments regarding the use of HSP90 inhibitors in cancer and how our new appreciation of the structural and functional heterogeneity of HSP90 invites a re-evaluation of how we discover and implement HSP90 therapeutics for disease treatment.

DOI: https://doi.org/10.1038/s41580-023-00640-9
Mol Cell Biol. 2023 Aug 02. 1-30

The Sequential Recruitments of Rab-GTPase Ypt1p and the NNS Complex onto pre-HAC1 mRNA Promote Its Nuclear Degradation in Baker's Yeast.

Sunirmal Paira, Anish Chakraborty, Biswadip Das.

  Induction of unfolded protein response involves activation of transcription factor Hac1p that is encoded by HAC1 pre-mRNA harboring an intron and a bipartite element (BE), which is subjected to nuclear mRNA decay by the nuclear exosome/Cbc1p-Tif4631p-dependent Exosome Targeting (CTEXT) complex. Using a combination of genetic and biochemical approaches, we demonstrate that a Rab-GTPase Ypt1p controls unfolded protein response signaling dynamics. This regulation relies on the nuclear localization of a small fraction of the cellular Ypt1p pool in the absence of endoplasmic reticulum (ER)-stress causing a strong association of the nuclear Ypt1p with pre-HAC1 mRNA that eventually promotes sequential recruitments of NNS, CTEXT, and the nuclear exosome onto this pre-mRNA. Recruitment of these decay factors onto pre-HAC1 mRNA is accompanied by its rapid nuclear decay that produces a precursor RNA pool lacking functional BE thereby causing its inefficient targeting to Ire1p foci leading to their diminished splicing and translation. ER stress triggers rapid relocalization of the nuclear pool of Ypt1p to the cytoplasm leading to its dissociation from pre-HAC1 mRNA thereby causing decreased recruitment of these decay factors to precursor HAC1 RNA leading to its diminished degradation. Reduced decay results in an increased abundance of pre-HAC1 mRNA with intact functional BE leading to its enhanced recruitment to Ire1p foci.

Keywords:  CBC1; CTEXT; HAC1; RRP6; UPR; Ypt1p; nuclear exosome

DOI:  https://doi.org/10.1080/10985549.2023.2227016
STAR Protoc. 2023 Jul 29. pii: S2666-1667(23)00425-2. [Epub ahead of print]4(3): 102458

Analyzing the topology of N-linked glycans by PNGase F accessibility assay.

Jingcheng Wang, Jin Ye.

  While N-glycans are synthesized in the lumens, some of them reach the cytosolic side of membranes through retro-translocation independent of endoplasmic-reticulum-associated degradation. Here, we present a protocol to measure the topology of N-glycans in a transmembrane protein, based on the principle that cytosolic but not luminal N-glycans are trimmed by PNGase F in the absence of detergent. We describe the procedures for this protocol consisting of microsome preparation from cells, PNGase F accessibility assay, and western blot analysis. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Keywords:  Cell Biology; Cell Separation/Fractionation; Cell-based Assays; Molecular Biology; Protein Biochemistry

DOI:  https://doi.org/10.1016/j.xpro.2023.102458
PLoS Biol. 2023 Aug 03. 21(8): e3002244

FBXO7/ntc and USP30 antagonistically set the ubiquitination threshold for basal mitophagy and provide a target for Pink1 phosphorylation in vivo.

Alvaro Sanchez-Martinez, Aitor Martinez, Alexander J Whitworth.

Functional analyses of genes linked to heritable forms of Parkinson's disease (PD) have revealed fundamental insights into the biological processes underpinning pathogenic mechanisms. Mutations in PARK15/FBXO7 cause autosomal recessive PD and FBXO7 has been shown to regulate mitochondrial homeostasis. We investigated the extent to which FBXO7 and its Drosophila orthologue, ntc, share functional homology and explored its role in mitophagy in vivo. We show that ntc mutants partially phenocopy Pink1 and parkin mutants and ntc overexpression supresses parkin phenotypes. Furthermore, ntc can modulate basal mitophagy in a Pink1- and parkin-independent manner by promoting the ubiquitination of mitochondrial proteins, a mechanism that is opposed by the deubiquitinase USP30. This basal ubiquitination serves as the substrate for Pink1-mediated phosphorylation that triggers stress-induced mitophagy. We propose that FBXO7/ntc works in equilibrium with USP30 to provide a checkpoint for mitochondrial quality control in basal conditions in vivo and presents a new avenue for therapeutic approaches.

DOI: https://doi.org/10.1371/journal.pbio.3002244
Proc Natl Acad Sci U S A. 2023 Aug 08. 120(32): e2304841120

Escherichia coli small heat shock protein IbpA plays a role in regulating the heat shock response by controlling the translation of σ32.

Tsukumi Miwa, Hideki Taguchi.

  Small heat shock proteins (sHsps) act as ATP-independent chaperones that prevent irreversible aggregate formation by sequestering denatured proteins. IbpA, an Escherichia coli sHsp, functions not only as a chaperone but also as a suppressor of its own expression through posttranscriptional regulation, contributing to negative feedback regulation. IbpA also regulates the expression of its paralog, IbpB, in a similar manner, but the extent to which IbpA regulates other protein expressions is unclear. We have identified that IbpA down-regulates the expression of many Hsps by repressing the translation of the heat shock transcription factor σ32. The IbpA regulation not only controls the σ32 level but also contributes to the shutoff of the heat shock response. These results revealed an unexplored role of IbpA to regulate heat shock response at a translational level, which adds an alternative layer for tightly controlled and rapid expression of σ32 on demand.

Keywords:  IbpA; heat shock response; rpoH; sHsp; small heat shock protein

DOI:  https://doi.org/10.1073/pnas.2304841120
J Biol Chem. 2023 Aug 01. pii: S0021-9258(23)02151-8. [Epub ahead of print] 105123

Mapping the MOB proteins' proximity network reveals a unique interaction between human MOB3C and the RNase P complex.

Islam E Elkholi, Jonathan Boulais, Marie-Pier Thibault, Hong-Duc Phan, Amélie Robert, Lien B Lai, Denis Faubert, Matthew J Smith, Venkat Gopalan, Jean-Franҫois Côté.

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions, and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification (BioID) approach to define the interactomes of all seven human MOB proteins in HeLa and HEK293 cell lines. We uncovered > 200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 out of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof-of-principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.

DOI: https://doi.org/10.1016/j.jbc.2023.105123
iScience. 2023 Jul 21. 26(7): 107180

Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

Hema Saranya Ilamathi, Sara Benhammouda, Amel Lounas, Khalid Al-Naemi, Justine Desrochers-Goyette, Matthew A Lines, François J Richard, Jackie Vogel, Marc Germain.

  Mitochondria are multifaceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) replication is a spatially regulated process essential for the maintenance of mitochondrial function, its defect causing mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is affected by mitochondrial dynamics: The absence of mitochondrial fusion is associated with mtDNA depletion whereas loss of mitochondrial fission causes the aggregation of mtDNA within abnormal structures termed mitobulbs. Here, we show that contact sites between mitochondria and ER sheets, the ER structure associated with protein synthesis, regulate mtDNA replication and distribution within mitochondrial networks. DRP1 loss or mutation leads to modified ER sheets and alters the interaction between ER sheets and mitochondria, disrupting RRBP1-SYNJ2BP interaction. Importantly, mtDNA distribution and replication were rescued by promoting ER sheets-mitochondria contact sites. Our work identifies the role of ER sheet-mitochondria contact sites in regulating mtDNA replication and distribution.

Keywords:  Biochemistry; Biological sciences; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2023.107180
Cell Rep. 2023 Jul 28. pii: S2211-1247(23)00903-8. [Epub ahead of print]42(8): 112892

Functional analogs of mammalian 4E-BPs reveal a role for TOR in global plant translation.

Yihan Dong, Ola Srour, Nina Lukhovitskaya, Joelle Makarian, Nicolas Baumberger, Oxana Galzitskaya, David Elser, Mikhail Schepetilnikov, Lyubov A Ryabova.

  Mammalian/mechanistic target of rapamycin (mTOR) regulates global protein synthesis through inactivation of eIF4E-binding proteins (m4E-BPs) in response to nutrient and energy availability. Until now, 4E-BPs have been considered as metazoan inventions, and how target of rapamycin (TOR) controls cap-dependent translation initiation in plants remains obscure. Here, we present short unstructured 4E-BP-like Arabidopsis proteins (4EBP1/4EBP2) that are non-homologous to m4E-BPs except for the eIF4E-binding motif and TOR phosphorylation sites. Unphosphorylated 4EBPs exhibit strong affinity toward eIF4Es and can inhibit formation of the cap-binding complex. Upon TOR activation, 4EBPs are phosphorylated, probably when bound directly to TOR, and likely relocated to ribosomes. 4EBPs can suppress a distinct set of mRNAs; 4EBP2 predominantly inhibits translation of core cell-cycle regulators CycB1;1 and CycD1;1, whereas 4EBP1 interferes with chlorophyll biosynthesis. Accordingly, 4EBP2 overexpression halts early seedling development, which is overcome by induction of Glc/Suc-TOR signaling. Thus, TOR regulates cap-dependent translation initiation by inactivating atypical 4EBPs in plants.

Keywords:  CP: Plants; Cap-dependent translation initiation; TOR phosphorylation targets; chlorophyll biosynthesis; cyclin B; cyclin D; eIF4E-binding proteins; eIF4E-type proteins; eIF4G-type proteins; translation control

DOI:  https://doi.org/10.1016/j.celrep.2023.112892
ACS Cent Sci. 2023 Jul 26. 9(7): 1269-1284

Proximity-Based Modalities for Biology and Medicine.

Xingui Liu, Alessio Ciulli.

Molecular proximity orchestrates biological function, and blocking existing proximities is an established therapeutic strategy. By contrast, strengthening or creating neoproximity with chemistry enables modulation of biological processes with high selectivity and has the potential to substantially expand the target space. A plethora of proximity-based modalities to target proteins via diverse approaches have recently emerged, opening opportunities for biopharmaceutical innovation. This Outlook outlines the diverse mechanisms and molecules based on induced proximity, including protein degraders, blockers, and stabilizers, inducers of protein post-translational modifications, and agents for cell therapy, and discusses opportunities and challenges that the field must address to mature and unlock translation in biology and medicine.

DOI: https://doi.org/10.1021/acscentsci.3c00395
J Biol Chem. 2023 Jul 29. pii: S0021-9258(23)02142-7. [Epub ahead of print] 105114

ER stress decreases exosome production through adiponectin/T-cadherin-dependent and -independent pathways.

Keita Fukuoka, Ryohei Mineo, Shunbun Kita, Shiro Fukuda, Tomonori Okita, Emi Kawada-Horitani, Masahito Iioka, Kohei Fujii, Keitaro Kawada, Yuya Fujishima, Hitoshi Nishizawa, Maeda Norikazu, Iichiro Shimomura.

  Exosomes, extracellular vesicles (EVs) produced within cells, mediate both the disposal of intracellular waste and communication with distant cells, and they are involved in a variety of disease processes. Although disease modifications of exosome cargos have been well studied, it has been poorly investigated how disease processes, such as endoplasmic reticulum (ER) stress, affect EV production. We previously reported that adiponectin, an adipocyte-secreted salutary factor, increases systemic exosome levels through T-cadherin-mediated enhancement of exosome biogenesis. In the present study, we demonstrated that adiponectin/T-cadherin-dependent EV production was susceptible to ER stress and that low-dose tunicamycin significantly reduced EV production in the presence, but not in the absence, of adiponectin. Moreover, pharmacological or genetic activation of IRE1α, a central regulator of ER stress, downregulated T-cadherin at the mRNA and protein levels as well as attenuated EV production. In addition, adiponectin/T-cadherin-independent EV production was attenuated under ER stress conditions. Repeated administration of tunicamycin to mice decreased circulating small EVs without decreasing tissue T-cadherin expression. Mechanistically, IRE1α activation by silencing of the XBP1 transcription factor upregulated the canonical IFN pathway and decreased EV production. The IFN pathway, when it was activated by polyinosinic-polycytidylic acid [poly(I:C)], also significantly attenuated EV production. Thus, we concluded that ER stress decreases exosome production through adiponectin/T-cadherin-dependent and -independent pathways.

Keywords:  Adiponectin; ER stress; Exosome; Extracellular vesicle; T-cadherin

DOI:  https://doi.org/10.1016/j.jbc.2023.105114
Nat Biotechnol. 2023 Aug 03.

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.

Kayvon Pedram, D Judy Shon, Gabrielle S Tender, Natalia R Mantuano, Jason J Northey, Kevin J Metcalf, Simon P Wisnovsky, Nicholas M Riley, Giovanni C Forcina, Stacy A Malaker, Angel Kuo, Benson M George, Caitlyn L Miller, Kerriann M Casey, José G Vilches-Moure, Michael J Ferracane, Valerie M Weaver, Heinz Läubli, Carolyn R Bertozzi.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

DOI: https://doi.org/10.1038/s41587-023-01840-6
Autophagy. 2023 Jul 30. 1-20

AI-based AlphaFold2 significantly expands the structural space of the autophagy pathway.

Nidhi Malhotra, Shantanu Khatri, Ajit Kumar, Akanksha Arun, Purba Daripa, Saman Fatihi, Sureshkumar Venkadesan, Niyati Jain, Lipi Thukral.

   ABBREVIATIONS: AF2: AlphaFold2; AF2-Mult: AlphaFold2 multimer; ATG: autophagy-related; CTD: C-terminal domain; ECTD: extreme C-terminal domain; FR: flexible region; MD: molecular dynamics; NTD: N-terminal domain; pLDDT: predicted local distance difference test; UBL: ubiquitin-like.

Keywords:  ATG7-ATG10 complex; AlphaFold2; autophagic interactome; autophagy; molecular dynamics simulations

DOI:  https://doi.org/10.1080/15548627.2023.2238578
Nat Commun. 2023 08 02. 14(1): 4624

BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling.

Jiaojiao Bai, Yuanyuan Zhou, Jianhang Sun, Kexin Chen, Yufang Han, Ranran Wang, Yanmin Zou, Mingshuo Du, Dongping Lu.

Pathogen-associated molecular patterns (PAMPs) trigger plant innate immunity that acts as the first line of inducible defense against pathogen infection. A receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) functions as a signaling hub immediately downstream of multiple pattern recognition receptors (PRRs). It is known that PLANT U-BOX PROTEIN 25 (PUB25) and PUB26 ubiquitinate BIK1 and mediate BIK1 degradation. However, how BIK1 homeostasis is maintained is not fully understood. Here, we show that two closely related ubiquitin ligases, RING DOMAIN LIGASE 1 (RGLG1) and RGLG2, preferentially associate with the hypo-phosphorylated BIK1 and promote the association of BIK1 with the co-receptor for several PRRs, BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). PUB25 interacts with RGLG2 and mediates its degradation. In turn, RGLG2 represses the ubiquitin ligase activity of PUB25. RGLG1/2 suppress PUB25-mediated BIK1 degradation, promote BIK1 protein accumulation, and positively regulate immune signaling in a ubiquitin ligase activity-dependent manner. Our work reveals how BIK1 homeostasis is maintained by the interplay of different ubiquitin ligases.

DOI: https://doi.org/10.1038/s41467-023-40364-0
Redox Biol. 2023 Jul 28. pii: S2213-2317(23)00234-3. [Epub ahead of print]65 102833

Loss of PERK function promotes ferroptosis by downregulating SLC7A11 (System Xc⁻) in colorectal cancer.

Krishan Kumar Saini, Priyank Chaturvedi, Abhipsa Sinha, Manish Pratap Singh, Muqtada Ali Khan, Ayushi Verma, Mushtaq Ahmad Nengroo, Saumya Ranjan Satrusal, Sanjeev Meena, Akhilesh Singh, Sameer Srivastava, Jayanta Sarkar, Dipak Datta.

  Ferroptosis, a genetically and biochemically distinct form of programmed cell death, is characterised by an iron-dependent accumulation of lipid peroxides. Therapy-resistant tumor cells display vulnerability toward ferroptosis. Endoplasmic Reticulum (ER) stress and Unfolded Protein Response (UPR) play a critical role in cancer cells to become therapy resistant. Tweaking the balance of UPR to make cancer cells susceptible to ferroptotic cell death could be an attractive therapeutic strategy. To decipher the emerging contribution of ER stress in the ferroptotic process, we observe that ferroptosis inducer RSL3 promotes UPR (PERK, ATF6, and IRE1α), along with overexpression of cystine-glutamate transporter SLC7A11 (System Xc-). Exploring the role of a particular UPR arm in modulating SLC7A11 expression and subsequent ferroptosis, we notice that PERK is selectively critical in inducing ferroptosis in colorectal carcinoma. PERK inhibition reduces ATF4 expression and recruitment to the promoter of SLC7A11 and results in its downregulation. Loss of PERK function not only primes cancer cells for increased lipid peroxidation but also limits in vivo colorectal tumor growth, demonstrating active signs of ferroptotic cell death in situ. Further, by performing TCGA data mining and using colorectal cancer patient samples, we demonstrate that the expression of PERK and SLC7A11 is positively correlated. Overall, our experimental data indicate that PERK is a negative regulator of ferroptosis and loss of PERK function sensitizes colorectal cancer cells to ferroptosis. Therefore, small molecule PERK inhibitors hold huge promise as novel therapeutics and their potential can be harnessed against the apoptosis-resistant condition.

Keywords:  Cancer; ER stress; Ferroptosis; PERK; SLC7A11; UPR

DOI:  https://doi.org/10.1016/j.redox.2023.102833
Cell. 2023 Jul 19. pii: S0092-8674(23)00733-X. [Epub ahead of print]

Structural basis for membrane-proximal proteolysis of substrates by ADAM10.

Colin H Lipper, Emily D Egan, Khal-Hentz Gabriel, Stephen C Blacklow.

  The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

Keywords:  APP; Alzheimer’s disease; Notch signaling; alpha-secretase; ectodomain shedding; metalloprotease; tetraspanin

DOI:  https://doi.org/10.1016/j.cell.2023.06.026
Nature. 2023 Aug 02.

cGAS-STING drives ageing-related inflammation and neurodegeneration.

Muhammet F Gulen, Natasha Samson, Alexander Keller, Marius Schwabenland, Chong Liu, Selene Glück, Vivek V Thacker, Lucie Favre, Bastien Mangeat, Lona J Kroese, Paul Krimpenfort, Marco Prinz, Andrea Ablasser.

Low-grade inflammation is a hallmark of old age and a central driver of ageing-associated impairment and disease1. Multiple factors can contribute to ageing-associated inflammation2; however, the molecular pathways that transduce aberrant inflammatory signalling and their impact in natural ageing remain unclear. Here we show that the cGAS-STING signalling pathway, which mediates immune sensing of DNA3, is a critical driver of chronic inflammation and functional decline during ageing. Blockade of STING suppresses the inflammatory phenotypes of senescent human cells and tissues, attenuates ageing-related inflammation in multiple peripheral organs and the brain in mice, and leads to an improvement in tissue function. Focusing on the ageing brain, we reveal that activation of STING triggers reactive microglial transcriptional states, neurodegeneration and cognitive decline. Cytosolic DNA released from perturbed mitochondria elicits cGAS activity in old microglia, defining a mechanism by which cGAS-STING signalling is engaged in the ageing brain. Single-nucleus RNA-sequencing analysis of microglia and hippocampi of a cGAS gain-of-function mouse model demonstrates that engagement of cGAS in microglia is sufficient to direct ageing-associated transcriptional microglial states leading to bystander cell inflammation, neurotoxicity and impaired memory capacity. Our findings establish the cGAS-STING pathway as a driver of ageing-related inflammation in peripheral organs and the brain, and reveal blockade of cGAS-STING signalling as a potential strategy to halt neurodegenerative processes during old age.

DOI: https://doi.org/10.1038/s41586-023-06373-1
Nat Commun. 2023 07 31. 14(1): 4594

Engineering tRNA abundances for synthetic cellular systems.

Akshay J Maheshwari, Jonathan Calles, Sean K Waterton, Drew Endy.

Routinizing the engineering of synthetic cells requires specifying beforehand how many of each molecule are needed. Physics-based tools for estimating desired molecular abundances in whole-cell synthetic biology are missing. Here, we use a colloidal dynamics simulator to make predictions for how tRNA abundances impact protein synthesis rates. We use rational design and direct RNA synthesis to make 21 synthetic tRNA surrogates from scratch. We use evolutionary algorithms within a computer aided design framework to engineer translation systems predicted to work faster or slower depending on tRNA abundance differences. We build and test the so-specified synthetic systems and find qualitative agreement between expected and observed systems. First principles modeling combined with bottom-up experiments can help molecular-to-cellular scale synthetic biology realize design-build-work frameworks that transcend tinker-and-test.

DOI: https://doi.org/10.1038/s41467-023-40199-9
Cell Rep. 2023 Aug 01. pii: S2211-1247(23)00891-4. [Epub ahead of print] 112880

Senescent cells form nuclear foci that contain the 26S proteasome.

Tomohiro Iriki, Hiroaki Iio, Shu Yasuda, Shun Masuta, Masakazu Kato, Hidetaka Kosako, Shoshiro Hirayama, Akinori Endo, Fumiaki Ohtake, Mako Kamiya, Yasuteru Urano, Yasushi Saeki, Jun Hamazaki, Shigeo Murata.

  The proteasome plays a central role in intracellular protein degradation. Age-dependent decline in proteasome activity is associated with cellular senescence and organismal aging; however, the mechanism by which the proteasome plays a role in senescent cells remains elusive. Here, we show that nuclear foci that contain the proteasome and exhibit liquid-like properties are formed in senescent cells. The formation of senescence-associated nuclear proteasome foci (SANPs) is dependent on ubiquitination and RAD23B, similar to previously known nuclear proteasome foci, but also requires proteasome activity. RAD23B knockdown suppresses SANP formation and increases mitochondrial activity, leading to reactive oxygen species production without affecting other senescence traits such as cell-cycle arrest and cell morphology. These findings suggest that SANPs are an important feature of senescent cells and uncover a mechanism by which the proteasome plays a role in senescent cells.

Keywords:  CP: Cell biology; CP: Molecular biology; cell senescence; liquid-liquid phase separation; mitochondria; nuclear body; proteasome; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2023.112880