bims-proteo 2024-08-18 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–08–18
forty-four papers selected by
Eric Chevet, INSERM

Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes Ubc6 and Ubc7 confer resistance to hygromycin B in Saccharomyces cerevisiae.
Sigma-1 receptor recruits LC3 mRNA to ER-associated omegasomes to promote localized LC3 translation enabling functional autophagy.
Mechanochemical forces regulate the composition and function of CAT tails.
Protein misfolding releases human Heat Shock Factor 1 (HSF1) from HSP70 latency control.
ER-phagy drives age-onset remodeling of endoplasmic reticulum structure-function and lifespan.
Optogenetic induction of mechanical muscle stress identifies myosin regulatory ubiquitin ligase NHL-1 in C. elegans.
Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity.
Identification of ERAD-dependent degrons for the endoplasmic reticulum lumen.
Insights into the interaction between UGGT, the gatekeeper of folding in the ER, and its partner, the selenoprotein SEP15.
OTUD6 deubiquitination of RPS7/eS7 on the free 40 S ribosome regulates global protein translation and stress.
Protein folding: From physics-chemical rules and cellular machineries of protein quality control to AI solutions.
P-NADs: PUX-based NAnobody degraders for ubiquitin-independent degradation of target proteins.
Conjugation of ATG8s to single membranes at a glance.
UPF3B modulates endoplasmic reticulum stress through interaction with inositol-requiring enzyme-1α.
Inter-compatibility of eukaryotic and Asgard archaea ribosome-translocon machineries.
Nuclear poly-glutamine aggregates rupture the nuclear envelope and hinder its repair.
Megakaryocyte maturation involves activation of the adaptive unfolded protein response.
Structural requirements for activity of Mind bomb1 in Notch signaling.
Interplay of PROTAC Complex Dynamics for Undruggable Targets: Insights into Ternary Complex Behavior and Linker Design.
The Ribosome Assembly Factor LSG1 Interacts with Vesicle-Associated Membrane Protein-Associated Proteins (VAPs).
Multi-tiered chemical proteomic maps of tryptoline acrylamide-protein interactions in cancer cells.
The importance of the location of the N-terminus in successful protein folding in vivo and in vitro.
The endolysosomal system in conventional and unconventional protein secretion.
Protein folding and quality control during nuclear transport.
The FAM114A proteins are adaptors for the recycling of Golgi enzymes.
Structural basis for transthiolation intermediates in the ubiquitin pathway.
Peptide-Mediated Small Molecule Lysosome-Targeting Chimeras for Targeted Degradation of Membrane and Intracellular Proteins.
Integrated stress response activator halofuginone protects mice from diabetes-like phenotypes.
A PIKfyve modulator combined with an integrated stress response inhibitor to treat lysosomal storage diseases.
Remodelling of the translatome controls diet and its impact on tumorigenesis.
Mitochondrial-derived compartments are multilamellar domains that encase membrane cargo and cytosol.
Rp3: Ribosome profiling-assisted proteogenomics improves coverage and confidence during microprotein discovery.
Degradation of STIM1 through FAM134B-mediated ER-phagy is potentially involved in cell proliferation.
Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma.
Redox-active chemical chaperones exhibiting promiscuous binding promote oxidative protein folding under condensed sub-millimolar conditions.
The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function.
Proximity analysis of native proteomes reveals phenotypic modifiers in a mouse model of autism and related neurodevelopmental conditions.
Protocol for the isolation and purification of endoplasmic reticulum-plasma membrane junctions from the mouse brain.
De novo design of allosterically switchable protein assemblies.
Dynamic interaction of REEP5-MFN1/2 enables mitochondrial hitchhiking on tubular ER.
Defective endomembrane dynamics in Rab27a deficiency impairs nucleic acid sensing and cytokine secretion in immune cells.
A spatiotemporally resolved atlas of mRNA decay in the C. elegans embryo reveals differential regulation of mRNA stability across stages and cell types.
Intestinal Nogo-B reduces GLP1 levels by binding to proglucagon on the endoplasmic reticulum to inhibit PCSK1 cleavage.
OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response.

MicroPubl Biol. 2024 ;2024

Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes Ubc6 and Ubc7 confer resistance to hygromycin B in Saccharomyces cerevisiae.

Sophia L Owutey, Katrina A Procuniar, Emmanuel Akoto, Jacob C Davis, Rachel M Vachon, LiLi F O'Malley, Hayden O Schneider, Philip J Smaldino, Jason D True, Ashley L Kalinski, Eric M Rubenstein.

Aberrant endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed through ER-associated degradation (ERAD) and INM-associated degradation (INMAD). We previously showed the Hrd1, Doa10, and Asi ERAD and INMAD ubiquitin ligases (E3s) in Saccharomyces cerevisiae confer resistance to hygromycin B, which distorts the ribosome decoding center. Here, we assessed the requirement of Ubc6 and Ubc7, the primary ERAD and INMAD ubiquitin-conjugating enzymes (E2s) for hygromycin B resistance. Loss of either E2 sensitized cells to hygromycin B, with UBC7 deletion having a greater impact, consistent with characterized roles for Ubc6 and Ubc7 in ER and INM protein quality control.

DOI: https://doi.org/10.17912/micropub.biology.001276
Cell Rep. 2024 Aug 10. pii: S2211-1247(24)00969-0. [Epub ahead of print]43(8): 114619

Sigma-1 receptor recruits LC3 mRNA to ER-associated omegasomes to promote localized LC3 translation enabling functional autophagy.

Jeffrey Knupp, Yu-Jie Chen, Emily Wang, Peter Arvan, Billy Tsai.

  Autophagosome formation initiated on the endoplasmic reticulum (ER)-associated omegasome requires LC3. Translational regulation of LC3 biosynthesis is unexplored. Here we demonstrate that LC3 mRNA is recruited to omegasomes by directly binding to the ER transmembrane Sigma-1 receptor (S1R). Cell-based and in vitro reconstitution experiments show that S1R interacts with the 3' UTR of LC3 mRNA and ribosomes to promote LC3 translation. Strikingly, the 3' UTR of LC3 is also required for LC3 protein lipidation, thereby linking the mRNA-3' UTR to LC3 function. An autophagy-defective S1R mutant responsible for amyotrophic lateral sclerosis cannot bind LC3 mRNA or induce LC3 translation. We propose a model wherein S1R de-represses LC3 mRNA via its 3' UTR at the ER, enabling LC3 biosynthesis and lipidation. Because several other LC3-related proteins use the same mechanism, our data reveal a conserved pathway for localized translation essential for autophagosome biogenesis with insights illuminating the molecular basis of a neurodegenerative disease.

Keywords:  ALS; CP: Cell biology; CP: Molecular biology; LC3; autophagy; lipidation; omegasome

DOI:  https://doi.org/10.1016/j.celrep.2024.114619
bioRxiv. 2024 Aug 04. pii: 2024.08.02.606406. [Epub ahead of print]

Mechanochemical forces regulate the composition and function of CAT tails.

Danish Khan, Ananya A Vinayak, Cole S Sitron, Onn Brandman.

  The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acids (CAT tails) in an mRNA-free, non-canonical elongation process. CAT tail composition includes Ala, Thr, and potentially other residues. The relationship between CAT tail composition and function has remained unknown. Using biochemical approaches in yeast, we discovered that mechanochemical forces on the NC regulate CAT tailing. We propose CAT tailing initially operates in an "extrusion mode" that increases NC lysine accessibility for on-ribosome ubiquitination. Thr in CAT tails enhances NC extrusion by preventing formation of polyalanine, which can form α-helices. After NC ubiquitylation, pulling forces on the NC switch CAT tailing to an Ala-only "release mode" which facilitates nascent chain release from large ribosomal subunits and NC degradation. Failure to switch from extrusion to release mode leads to accumulation of NCs on large ribosomal subunits and proteotoxic aggregation of Thr-rich CAT tails.

Keywords:  CAT tails; mechanochemistry; protein folding; protein quality control; ribosome; ribosome stalling; ribosome-associated quality control (RQC); translation

DOI:  https://doi.org/10.1101/2024.08.02.606406
J Mol Biol. 2024 Aug 07. pii: S0022-2836(24)00349-8. [Epub ahead of print] 168740

Protein misfolding releases human Heat Shock Factor 1 (HSF1) from HSP70 latency control.

Michela Ciccarelli, Claes Andréasson.

  Heat shock factor 1 (HSF1) responds to stress to mount the heat shock response (HSR), a conserved transcriptional program that allows cells to maintain proteostasis by upregulating heat shock proteins (HSPs). The homeostatic stress regulation of HSF1 plays a key role in human physiology and health but its mechanism has remained difficult to pinpoint. Recent work in the budding yeast model has implicated stress-inducible chaperones of the HSP70 family as direct negative regulators of HSF1 activity. Here, we have investigated the latency control and activation of human HSF1 by HSP70 and misfolded proteins. Purified oligomeric HSF1-HSP70 (HSPA1A) complexes exhibited basal DNA binding activity that was inhibited by increasing the levels of HSP70 and, importantly, misfolded proteins reverted the inhibitory effect. Using site-specific UV photo-crosslinking, we monitored HSP70-HSF1 complexes in HEK293T cells. While HSF1 was bound by the substrate binding domain of HSP70 in unstressed cells, activation of HSF1 by heat shock as well as by inducing the misfolding of newly synthesized proteins resulted in release of HSF1 from the chaperone. Taken our results together, we conclude that latent HSF1 populate dynamic complexes with HSP70, which are sensitive to increased levels of misfolded proteins that compete for binding to the HSP70 substrate binding domain. Thus, human HSF1 is activated by various stress conditions that all titrate available HSP70.

Keywords:  Proteostasis; azetidine-2-carboxylic acid; chaperone titration; heat shock; heat shock factor 1; heat shock protein 70; heat shock response; protein misfolding

DOI:  https://doi.org/10.1016/j.jmb.2024.168740
bioRxiv. 2024 Aug 08. pii: 2024.08.07.607085. [Epub ahead of print]

ER-phagy drives age-onset remodeling of endoplasmic reticulum structure-function and lifespan.

Ekf Donahue, N L Hepowit, B Keuchel, A G Mulligan, D J Johnson, M Ellisman, R Arrojo E Drigo, J MacGurn, K Burkewitz.

The endoplasmic reticulum (ER) comprises an array of structurally distinct subdomains, each with characteristic functions. While altered ER-associated processes are linked to age-onset pathogenesis, whether shifts in ER morphology underlie these functional changes is unclear. We report that ER remodeling is a conserved feature of the aging process in models ranging from yeast to C. elegans and mammals. Focusing on C. elegans as an exemplar of metazoan aging, we find that as animals age, ER mass declines in virtually all tissues and ER morphology shifts from rough sheets to tubular ER. The accompanying large-scale shifts in proteomic composition correspond to the ER turning from protein synthesis to lipid metabolism. To drive this substantial remodeling, ER-phagy is activated early in adulthood, promoting turnover of rough ER in response to rises in luminal protein-folding burden and reduced global protein synthesis. Surprisingly, ER remodeling is a pro-active and protective response during aging, as ER-phagy impairment limits lifespan in yeast and diverse lifespan-extending paradigms promote profound remodeling of ER morphology even in young animals. Altogether our results reveal ER-phagy and ER morphological dynamics as pronounced, underappreciated mechanisms of both normal aging and enhanced longevity.

DOI: https://doi.org/10.1101/2024.08.07.607085
Nat Commun. 2024 Aug 11. 15(1): 6879

Optogenetic induction of mechanical muscle stress identifies myosin regulatory ubiquitin ligase NHL-1 in C. elegans.

Carl Elias Kutzner, Karen Carolyn Bauer, Jan-Wilm Lackmann, Richard James Acton, Anwesha Sarkar, Wojciech Pokrzywa, Thorsten Hoppe.

Mechanical stress during muscle contraction is a constant threat to proteome integrity. However, there is a lack of experimental systems to identify critical proteostasis regulators under mechanical stress conditions. Here, we present the transgenic Caenorhabditis elegans model OptIMMuS (Optogenetic Induction of Mechanical Muscle Stress) to study changes in the proteostasis network associated with mechanical forces. Repeated blue light exposure of a muscle-expressed Chlamydomonas rheinhardii channelrhodopsin-2 variant results in sustained muscle contraction and mechanical stress. Using OptIMMuS, combined with proximity labeling and mass spectrometry, we identify regulators that cooperate with the myosin-directed chaperone UNC-45 in muscle proteostasis. One of these is the TRIM E3 ligase NHL-1, which interacts with UNC-45 and muscle myosin in genetic epistasis and co-immunoprecipitation experiments. We provide evidence that the ubiquitylation activity of NHL-1 regulates myosin levels and functionality under mechanical stress. In the future, OptIMMuS will help to identify muscle-specific proteostasis regulators of therapeutic relevance.

DOI: https://doi.org/10.1038/s41467-024-51069-3
PLoS One. 2024 ;19(8): e0303008

Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity.

Leeran B Dublin-Ryan, Ankan K Bhadra, Heather L True.

The nascent polypeptide-associate complex (NAC) is a heterodimeric chaperone complex that binds near the ribosome exit tunnel and is the first point of chaperone contact for newly synthesized proteins. Deletion of the NAC induces embryonic lethality in many multi-cellular organisms. Previous work has shown that the deletion of the NAC rescues cells from prion-induced cytotoxicity. This counterintuitive result led us to hypothesize that NAC disruption would improve viability in cells expressing human misfolding proteins. Here, we show that NAC disruption improves viability in cells expressing expanded polyglutamine and also leads to delayed and reduced aggregation of expanded polyglutamine and changes in polyglutamine aggregate morphology. Moreover, we show that NAC disruption leads to changes in de novo yeast prion induction. These results indicate that the NAC plays a critical role in aggregate organization as a potential therapeutic target in neurodegenerative disorders.

DOI: https://doi.org/10.1371/journal.pone.0303008
bioRxiv. 2024 Aug 05. pii: 2023.06.21.546000. [Epub ahead of print]

Identification of ERAD-dependent degrons for the endoplasmic reticulum lumen.

Rachel Sharninghausen, Jiwon Hwang, Devon D Dennison, Ryan D Baldridge.

Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae , we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the ERAD system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly-conserved ERAD system.

DOI: https://doi.org/10.1101/2023.06.21.546000
Proc Natl Acad Sci U S A. 2024 Aug 20. 121(34): e2315009121

Insights into the interaction between UGGT, the gatekeeper of folding in the ER, and its partner, the selenoprotein SEP15.

Robert V Williams, Kevin P Guay, Owen A Hurlbut Lesk, Eugenia M Clerico, Daniel N Hebert, Lila M Gierasch.

  The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15.

Keywords:  SEP15; UGGT; endoplasmic reticulum; protein folding

DOI:  https://doi.org/10.1073/pnas.2315009121
Nat Commun. 2024 Aug 11. 15(1): 6873

OTUD6 deubiquitination of RPS7/eS7 on the free 40 S ribosome regulates global protein translation and stress.

Sammy Villa, Pankaj Dwivedi, Aaron Stahl, Trent Hinkle, Christopher M Rose, Donald S Kirkpatrick, Seth M Tomchik, Vishva M Dixit, Fred W Wolf.

Ribosomes are regulated by evolutionarily conserved ubiquitination/deubiquitination events. We uncover the role of the deubiquitinase OTUD6 in regulating global protein translation through deubiquitination of the RPS7/eS7 subunit on the free 40 S ribosome in vivo in Drosophila. Coimmunoprecipitation and enrichment of monoubiquitinated proteins from catalytically inactive OTUD6 flies reveal RPS7 as the ribosomal substrate. The 40 S protein RACK1 and E3 ligases CNOT4 and RNF10 function upstream of OTUD6 to regulate alkylation stress. OTUD6 interacts with RPS7 specifically on the free 40 S, and not on 43 S/48 S initiation complexes or the translating ribosome. Global protein translation levels are bidirectionally regulated by OTUD6 protein abundance. OTUD6 protein abundance is physiologically regulated in aging and in response to translational and alkylation stress. Thus, OTUD6 may promote translation initiation, the rate limiting step in protein translation, by titering the amount of 40 S ribosome that recycles.

DOI: https://doi.org/10.1038/s41467-024-51284-y
Proc Natl Acad Sci U S A. 2024 Aug 20. 121(34): e2411135121

Protein folding: From physics-chemical rules and cellular machineries of protein quality control to AI solutions.

Ulyana Shimanovich, F Ulrich Hartl.

DOI: https://doi.org/10.1073/pnas.2411135121
Heliyon. 2024 Jul 30. 10(14): e34487

P-NADs: PUX-based NAnobody degraders for ubiquitin-independent degradation of target proteins.

Jun Wang, Georgy Chistov, Junrui Zhang, Brandon Huntington, Israa Salem, Anandsukeerthi Sandholu, Stefan T Arold.

  Targeted protein degradation (TPD) allows cells to maintain a functional proteome and to rapidly adapt to changing conditions. Methods that repurpose TPD for the deactivation of specific proteins have demonstrated significant potential in therapeutic and research applications. Most of these methods are based on proteolysis targeting chimaeras (PROTACs) which link the protein target to an E3 ubiquitin ligase, resulting in the ubiquitin-based degradation of the target protein. In this study, we introduce a method for ubiquitin-independent TPD based on nanobody-conjugated plant ubiquitin regulatory X domain-containing (PUX) adaptor proteins. We show that the PUX-based NAnobody Degraders (P-NADs) can unfold a target protein through the Arabidopsis and human orthologues of the CDC48 unfoldase without the need for ubiquitination or initiating motifs. We demonstrate that P-NAD plasmids can be transfected into a human cell line, where the produced P-NADs use the endogenous CDC48 machinery for ubiquitin-independent TPD of a 143 kDa multidomain protein. Thus, P-NADs pave the road for ubiquitin-independent therapeutic TPD approaches. In addition, the modular P-NAD design combined with in vitro and cellular assays provide a versatile platform for elucidating functional aspects of CDC48-based TPD in plants and animals.

Keywords:  CDC48; PROTAC; Plant ubiquitin regulatory X domain-containing; Targeted protein degradation; p97

DOI:  https://doi.org/10.1016/j.heliyon.2024.e34487
J Cell Sci. 2024 Aug 01. pii: jcs261031. [Epub ahead of print]137(15):

Conjugation of ATG8s to single membranes at a glance.

Carmen Figueras-Novoa, Lewis Timimi, Elena Marcassa, Rachel Ulferts, Rupert Beale.

  Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.

Keywords:  ATG8ylation; Autophagy; CASM; STIL; TECPR1; V-ATPase; VAIL

DOI:  https://doi.org/10.1242/jcs.261031
Cell Death Dis. 2024 Aug 13. 15(8): 587

UPF3B modulates endoplasmic reticulum stress through interaction with inositol-requiring enzyme-1α.

XingSheng Sun, Ruqin Lin, Xinxia Lu, Zhikai Wu, Xueying Qi, Tianqing Jiang, Jun Jiang, Peiqiang Mu, Qingmei Chen, Jikai Wen, Yiqun Deng.

The unfolded protein response (UPR) is a conserved and adaptive intracellular pathway that relieves the endoplasmic reticulum (ER) stress by activating ER transmembrane stress sensors. As a consequence of ER stress, the inhibition of nonsense-mediated mRNA decay (NMD) is due to an increase in the phosphorylation of eIF2α, which has the effect of inhibiting translation. However, the role of NMD in maintaining ER homeostasis remains unclear. In this study, we found that the three NMD factors, up-frameshift (UPF)1, UPF2, or UPF3B, were required to negate the UPR. Among these three NMD factors, only UPF3B interacted with inositol-requiring enzyme-1α (IRE1α). This interaction inhibited the kinase activity of IRE1α, abolished autophosphorylation, and reduced IRE1α clustering for ER stress. BiP and UPF3B jointly control the activation of IRE1α on both sides of the ER membrane. Under stress conditions, the phosphorylation of UPF3B was increased and the phosphorylated sites were identified. Both the UPF3BY160D genetic mutation and phosphorylation at Thr169 of UPF3B abolished its interaction with IRE1α and UPF2, respectively, leading to activation of ER stress and NMD dysfunction. Our study reveals a key physiological role for UPF3B in the reciprocal regulatory relationship between NMD and ER stress.

DOI: https://doi.org/10.1038/s41419-024-06973-3
J Biol Chem. 2024 Aug 09. pii: S0021-9258(24)02174-4. [Epub ahead of print] 107673

Inter-compatibility of eukaryotic and Asgard archaea ribosome-translocon machineries.

Isaac Carilo, Yosuke Senju, Takeshi Yokoyama, Robert C Robinson.

  In all domains of life, the ribosome-translocon complex inserts nascent transmembrane proteins into, and processes and transports signal peptide-containing proteins across, membranes. Eukaryotic translocons are anchored in the endoplasmic reticulum, while the prokaryotic complexes reside in cell membranes. Phylogenetic analyses indicate inheritance of eukaryotic Sec61/OST/TRAP translocon subunits from an Asgard archaea ancestor. However, the mechanism for translocon migration from a peripheral membrane to an internal cellular compartment (the proto-endoplasmic reticulum) during eukaryogenesis is unknown. Here we show compatibility between the eukaryotic ribosome-translocon complex and Asgard signal peptides and transmembrane proteins. We find that Asgard translocon proteins from Candidatus Prometheoarchaeum syntrophicum strain MK-D1, a Lokiarchaeon confirmed to contain no internal cellular membranes, are targeted to the eukaryotic endoplasmic reticulum on ectopic expression. Furthermore, we show that the cytoplasmic domain of MK-D1 OST1 (ribophorin I) can interact with eukaryotic ribosomes. Our data indicate that the location of existing ribosome-translocon complexes, at the protein level, determines the future placement of yet to be translated translocon subunits. This principle predicts that during eukaryogenesis, under positive selection pressure, the relocation of a few translocon complexes to the proto-endoplasmic reticulum will have contributed to propagating the new translocon location, leading to their loss from the cell membrane.

Keywords:  Asgard archaea; Evolution; OST complex; Sec61; TRAP complex; X-ray crystallography; endoplasmic reticulum; translocon

DOI:  https://doi.org/10.1016/j.jbc.2024.107673
J Cell Biol. 2024 Nov 04. pii: e202307142. [Epub ahead of print]223(11):

Nuclear poly-glutamine aggregates rupture the nuclear envelope and hinder its repair.

Giel Korsten, Miriam Osinga, Robin A Pelle, Albert K Serweta, Baukje Hoogenberg, Harm H Kampinga, Lukas C Kapitein.

Huntington's disease (HD) is caused by a polyglutamine expansion of the huntingtin protein, resulting in the formation of polyglutamine aggregates. The mechanisms of toxicity that result in the complex HD pathology remain only partially understood. Here, we show that nuclear polyglutamine aggregates induce nuclear envelope (NE) blebbing and ruptures that are often repaired incompletely. These ruptures coincide with disruptions of the nuclear lamina and lead to lamina scar formation. Expansion microscopy enabled resolving the ultrastructure of nuclear aggregates and revealed polyglutamine fibrils sticking into the cytosol at rupture sites, suggesting a mechanism for incomplete repair. Furthermore, we found that NE repair factors often accumulated near nuclear aggregates, consistent with stalled repair. These findings implicate nuclear polyQ aggregate-induced loss of NE integrity as a potential contributing factor to Huntington's disease and other polyglutamine diseases.

DOI: https://doi.org/10.1083/jcb.202307142
Genes Cells. 2024 Aug 13.

Megakaryocyte maturation involves activation of the adaptive unfolded protein response.

M Faiz, M L Kalev-Zylinska, C Dunstan-Harrison, D C Singleton, M P Hay, E C Ledgerwood.

  Endoplasmic reticulum stress triggers the unfolded protein response (UPR) to promote cell survival or apoptosis. Transient endoplasmic reticulum stress activation has been reported to trigger megakaryocyte production, and UPR activation has been reported as a feature of megakaryocytic cancers. However, the role of UPR signaling in megakaryocyte biology is not fully understood. We studied the involvement of UPR in human megakaryocytic differentiation using PMA (phorbol 12-myristate 13-acetate)-induced maturation of megakaryoblastic cell lines and thrombopoietin-induced differentiation of human peripheral blood-derived progenitors. Our results demonstrate that an adaptive UPR is a feature of megakaryocytic differentiation and that this response is not associated with ER stress-induced apoptosis. Differentiation did not alter the response to the canonical endoplasmic reticulum stressors DTT or thapsigargin. However, thapsigargin, but not DTT, inhibited differentiation, consistent with the involvement of Ca2+ signaling in megakaryocyte differentiation.

Keywords:  IRE1α; XBP1; calcium; endoplasmic reticulum (ER) stress; megakaryopoiesis; unfolded protein response (UPR)

DOI:  https://doi.org/10.1111/gtc.13151
Structure. 2024 Jul 26. pii: S0969-2126(24)00274-0. [Epub ahead of print]

Structural requirements for activity of Mind bomb1 in Notch signaling.

Ruili Cao, Oren Gozlan, Alina Airich, Lena Tveriakhina, Haixia Zhou, Hanjie Jiang, Philip A Cole, Jon C Aster, Thomas Klein, David Sprinzak, Stephen C Blacklow.

  Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2∼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region (ccRING3) drive MIB1 dimerization and that MIB1 ubiquitin transfer activity relies solely on ccRING3. We report X-ray crystal structures of a UbcH5B-ccRING3 complex and the ANK domain. Directly tethering the MIB1 N-terminal region to ccRING3 forms a minimal MIB1 protein sufficient to induce a Notch response in receiver cells and rescue mib knockout phenotypes in flies. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.

Keywords:  Delta; Jagged; MIB1; Notch signaling; Notch1; X-ray crystallography; endocytosis

DOI:  https://doi.org/10.1016/j.str.2024.07.011
ACS Med Chem Lett. 2024 Aug 08. 15(8): 1306-1318

Interplay of PROTAC Complex Dynamics for Undruggable Targets: Insights into Ternary Complex Behavior and Linker Design.

Harish Kumar, Masilamani Elizabeth Sobhia.

Protein degraders, such as bifunctional proteolysis-targeting chimeras (PROTACs), selectively eliminate target proteins by leveraging the natural protein degradation machinery. PROTACs bridge the target protein with an E3 ligase, which induces ubiquitination and degradation. Investigating ternary complex structures elucidates the molecular mechanisms of their formation and degradation. This study examines the binding dynamics of E3 ligases (VHL, CRBN, and cIAP) with proteins of interest, focusing on dynamics, cooperativity, selectivity, linker length, and PROTAC conformations. The influence of interface residues and linker lengths on specific conformations for target proteins and E3 ligases is highlighted. Utilizing molecular dynamics and steered molecular dynamics simulations, the study provides comprehensive parameters on the behavior and stability of diverse ternary complexes. These insights are crucial for designing PROTACs targeting disease-causing proteins and advancing the development of degradable ternary complexes for therapeutic applications.

DOI: https://doi.org/10.1021/acsmedchemlett.4c00189
Mol Cell Biol. 2024 Aug 12. 1-13

The Ribosome Assembly Factor LSG1 Interacts with Vesicle-Associated Membrane Protein-Associated Proteins (VAPs).

Putri Sutjita, Sharmishtha Musalgaonkar, Jeffrey Recchia-Rife, Lisa Huang, Blerta Xhemalce, Arlen W Johnson.

  LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum. VAPs interact with a large host of proteins which contain FFAT motifs (two phenylalanines (FF) in an acidic tract) and are involved in many cellular functions including membrane traffic and regulation of lipid transport. Here, we show that human LSG1 binds to VAPs via a noncanonical FFAT-like motif. Deletion of this motif specifically disrupts the localization of LSG1 to the ER, without perturbing LSG1-dependent recycling of NMD3 in cells or modulation of LSG1 GTPase activity in vitro.

Keywords:  LSG1; Ribosome biogenesis; VAP; vesicle-associated membrane protein-associated protein

DOI:  https://doi.org/10.1080/10985549.2024.2384600
Nat Chem. 2024 Aug 13.

Multi-tiered chemical proteomic maps of tryptoline acrylamide-protein interactions in cancer cells.

Evert Njomen, Rachel E Hayward, Kristen E DeMeester, Daisuke Ogasawara, Melissa M Dix, Tracey Nguyen, Paige Ashby, Gabriel M Simon, Stuart L Schreiber, Bruno Melillo, Benjamin F Cravatt.

Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds-termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells.

DOI: https://doi.org/10.1038/s41557-024-01601-1
Proc Natl Acad Sci U S A. 2024 Aug 20. 121(34): e2321999121

The importance of the location of the N-terminus in successful protein folding in vivo and in vitro.

Natalie R Dall, Carolina A T F Mendonça, Héctor L Torres Vera, Susan Marqusee.

  Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.

Keywords:  aggregation; circular permutation; cotranslational folding; protein folding

DOI:  https://doi.org/10.1073/pnas.2321999121
J Cell Biol. 2024 Sep 02. pii: e202404152. [Epub ahead of print]223(9):

The endolysosomal system in conventional and unconventional protein secretion.

Eloïse Néel, Marioara Chiritoiu-Butnaru, William Fargues, Morgane Denus, Maëlle Colladant, Aurore Filaquier, Sarah E Stewart, Sylvain Lehmann, Chiara Zurzolo, David C Rubinsztein, Philippe Marin, Marie-Laure Parmentier, Julien Villeneuve.

Most secreted proteins are transported through the "conventional" endoplasmic reticulum-Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or "unconventional" secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes.

DOI: https://doi.org/10.1083/jcb.202404152
Curr Opin Cell Biol. 2024 Aug 13. pii: S0955-0674(24)00086-3. [Epub ahead of print]90 102407

Protein folding and quality control during nuclear transport.

Sunanda Mallik, Dylan Poch, Sophia Burick, Christian Schlieker.

The spatial separation of protein synthesis from the compartmental destiny of proteins led to the evolution of transport systems that are efficient and yet highly specific. Co-translational transport has emerged as a strategy to avoid cytosolic aggregation of folding intermediates and the need for energy-consuming unfolding strategies to enable transport through narrow conduits connecting compartments. While translation and compartmental translocation are at times tightly coordinated, we know very little about the temporal coordination of translation, protein folding, and nuclear import. Here, we consider the implications of co-translational engagement of nuclear import machinery. We propose that the dynamic interplay of karyopherins and intrinsically disordered nucleoporins create a favorable protein folding environment for cargo en route to the nuclear compartment while maintaining a barrier function of the nuclear pore complex. Our model is discussed in the context of neurological disorders that are tied to defects in nuclear transport and protein quality control.

DOI: https://doi.org/10.1016/j.ceb.2024.102407
J Cell Sci. 2024 Aug 12. pii: jcs.262160. [Epub ahead of print]

The FAM114A proteins are adaptors for the recycling of Golgi enzymes.

Lawrence G Welch, Nadine Muschalik, Sean Munro.

  Golgi resident enzymes remain in place whilst their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. We used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

Keywords:   Drosophila ; COPI; Glycosylation; Golgi; Recycling

DOI:  https://doi.org/10.1242/jcs.262160
Nature. 2024 Aug 14.

Structural basis for transthiolation intermediates in the ubiquitin pathway.

Tomasz Kochańczyk, Zachary S Hann, Michaelyn C Lux, Avelyn Mae V Delos Reyes, Cheng Ji, Derek S Tan, Christopher D Lima.

Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins1-3. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters4-6. How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1-Ub(T)-E2 and E2-Ub(T)-E3HECT complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.

DOI: https://doi.org/10.1038/s41586-024-07828-9
J Med Chem. 2024 Aug 15.

Peptide-Mediated Small Molecule Lysosome-Targeting Chimeras for Targeted Degradation of Membrane and Intracellular Proteins.

Shuqiang Chen, Wei Wang, Zhipeng Chen, Ruyan Li, Zhe Wu, Guoqiang Dong, Chunquan Sheng.

Targeted protein degradation through the lysosomal pathway has attracted increasing attention and expanded the scope of degradable proteins. However, the endogenous lysosomal degradation strategies are mainly based on antibodies or nanobodies. Effective small molecule lysosomal degraders are still rather rare. Herein, a new lysosomal degradation approach, termed peptide-mediated small molecule lysosome-targeting chimeras (PSMLTACs), was developed by the incorporation of small molecule ligands with a lysosome-sorting NPGY motif containing the cell-penetrating peptide. PSMLTACs were successfully applied to degrade both membrane and intracellular targets. In particular, the PSMLTAC strategy demonstrated higher degradation efficiency on membrane target PD-L1 and intracellular target PDEδ than corresponding PROTAC degraders. Taken together, this proof-of-concept provides a convenient and effective strategy for targeted protein degradation.

DOI: https://doi.org/10.1021/acs.jmedchem.4c01449
J Cell Biol. 2024 Oct 07. pii: e202405175. [Epub ahead of print]223(10):

Integrated stress response activator halofuginone protects mice from diabetes-like phenotypes.

Shashank Rai, Maria Szaruga, Aleksandra P Pitera, Anne Bertolotti.

The integrated stress response (ISR) is a vital signaling pathway initiated by four kinases, PERK, GCN2, HRI and PKR, that ensure cellular resilience and protect cells from challenges. Here, we investigated whether increasing ISR signaling could rescue diabetes-like phenotypes in a mouse model of diet-induced obesity (DIO). We show that the orally available and clinically approved GCN2 activator halofuginone (HF) can activate the ISR in mouse tissues. We found that daily oral administration of HF increases glucose tolerance whilst reducing weight gain, insulin resistance, and serum insulin in DIO mice. Conversely, the ISR inhibitor GSK2656157, used at low doses to optimize its selectivity, aggravates glucose intolerance in DIO mice. Whilst loss of function mutations in mice and humans have revealed that PERK is the essential ISR kinase that protects from diabetes, our work demonstrates the therapeutic value of increasing ISR signaling by activating the related kinase GCN2 to reduce diabetes phenotypes in a DIO mouse model.

DOI: https://doi.org/10.1083/jcb.202405175
Proc Natl Acad Sci U S A. 2024 Aug 20. 121(34): e2320257121

A PIKfyve modulator combined with an integrated stress response inhibitor to treat lysosomal storage diseases.

William C Hou, Lynée A Massey, Derek Rhoades, Yin Wu, Wen Ren, Chiara Frank, Herman S Overkleeft, Jeffrey W Kelly.

  Lysosomal degradation pathways coordinate the clearance of superfluous and damaged cellular components. Compromised lysosomal degradation is a hallmark of many degenerative diseases, including lysosomal storage diseases (LSDs), which are caused by loss-of-function mutations within both alleles of a lysosomal hydrolase, leading to lysosomal substrate accumulation. Gaucher's disease, characterized by <15% of normal glucocerebrosidase function, is the most common LSD and is a prominent risk factor for developing Parkinson's disease. Here, we show that either of two structurally distinct small molecules that modulate PIKfyve activity, identified in a high-throughput cellular lipid droplet clearance screen, can improve glucocerebrosidase function in Gaucher patient-derived fibroblasts through an MiT/TFE transcription factor that promotes lysosomal gene translation. An integrated stress response (ISR) antagonist used in combination with a PIKfyve modulator further improves cellular glucocerebrosidase activity, likely because ISR signaling appears to also be slightly activated by treatment by either small molecule at the higher doses employed. This strategy of combining a PIKfyve modulator with an ISR inhibitor improves mutant lysosomal hydrolase function in cellular models of additional LSD.

Keywords:  Gaucher’s disease; PIKfyve; Parkinson’s disease; integrated stress response; lysosomal storage disease

DOI:  https://doi.org/10.1073/pnas.2320257121
Nature. 2024 Aug 14.

Remodelling of the translatome controls diet and its impact on tumorigenesis.

Haojun Yang, Vincenzo Andrea Zingaro, James Lincoff, Harrison Tom, Satoshi Oikawa, Juan A Oses-Prieto, Quinn Edmondson, Ian Seiple, Hardik Shah, Shingo Kajimura, Alma L Burlingame, Michael Grabe, Davide Ruggero.

Fasting is associated with a range of health benefits1-6. How fasting signals elicit changes in the proteome to establish metabolic programmes remains poorly understood. Here we show that hepatocytes selectively remodel the translatome while global translation is paradoxically downregulated during fasting7,8. We discover that phosphorylation of eukaryotic translation initiation factor 4E (P-eIF4E) is induced during fasting. We show that P-eIF4E is responsible for controlling the translation of genes involved in lipid catabolism and the production of ketone bodies. Inhibiting P-eIF4E impairs ketogenesis in response to fasting and a ketogenic diet. P-eIF4E regulates those messenger RNAs through a specific translation regulatory element within their 5' untranslated regions (5' UTRs). Our findings reveal a new signalling property of fatty acids, which are elevated during fasting. We found that fatty acids bind and induce AMP-activated protein kinase (AMPK) kinase activity that in turn enhances the phosphorylation of MAP kinase-interacting protein kinase (MNK), the kinase that phosphorylates eIF4E. The AMPK-MNK-eIF4E axis controls ketogenesis, revealing a new lipid-mediated kinase signalling pathway that links ketogenesis to translation control. Certain types of cancer use ketone bodies as an energy source9,10 that may rely on P-eIF4E. Our findings reveal that on a ketogenic diet, treatment with eFT508 (also known as tomivosertib; a P-eIF4E inhibitor) restrains pancreatic tumour growth. Thus, our findings unveil a new fatty acid-induced signalling pathway that activates selective translation, which underlies ketogenesis and provides a tailored diet intervention therapy for cancer.

DOI: https://doi.org/10.1038/s41586-024-07781-7
J Cell Biol. 2024 Nov 04. pii: e202307035. [Epub ahead of print]223(11):

Mitochondrial-derived compartments are multilamellar domains that encase membrane cargo and cytosol.

Zachary N Wilson, Matt West, Alyssa M English, Greg Odorizzi, Adam L Hughes.

Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here, we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains.

DOI: https://doi.org/10.1083/jcb.202307035
Nat Commun. 2024 Aug 09. 15(1): 6839

Rp3: Ribosome profiling-assisted proteogenomics improves coverage and confidence during microprotein discovery.

Eduardo Vieira de Souza, Angie L Bookout, Christopher A Barnes, Brendan Miller, Pablo Machado, Luiz A Basso, Cristiano V Bizarro, Alan Saghatelian.

There has been a dramatic increase in the identification of non-canonical translation and a significant expansion of the protein-coding genome. Among the strategies used to identify unannotated small Open Reading Frames (smORFs) that encode microproteins, Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple genomic sites are removed since they cannot be unambiguously assigned to a specific genomic location. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of multi-mapping alignments, such that smORFs residing in these regions cannot be identified by Ribo-Seq. Moreover, it has been challenging to identify protein evidence for Ribo-Seq. To solve this, we developed Rp3, a pipeline that integrates proteogenomics and Ribosome profiling to provide unambiguous evidence for a subset of microproteins missed by current Ribo-Seq pipelines. Here, we show that Rp3 maximizes proteomics detection and confidence of microprotein-encoding smORFs.

DOI: https://doi.org/10.1038/s41467-024-50301-4
J Biol Chem. 2024 Aug 09. pii: S0021-9258(24)02175-6. [Epub ahead of print] 107674

Degradation of STIM1 through FAM134B-mediated ER-phagy is potentially involved in cell proliferation.

Hiroaki Kajiho, Toshiaki Sakisaka.

  Autophagy is classified as non-selective or selective depending on the types of degrading substrates. Endoplasmic reticulum (ER)-phagy is a form of selective autophagy for transporting the ER-resident proteins to autolysosomes. FAM134B, a member of the family with sequence similarity 134, is a well-known ER-phagy receptor. Dysfunction of FAM134B results in several diseases including viral infection, inflammation, neurodegenerative disorder and cancer, indicating that FAM134B has crucial roles in various kinds of intracellular functions. However, how FAM134B-mediated ER-phagy regulates intracellular functions is not well understood. In this study, we found that FAM134B knockdown in mammalian cells accelerated cell proliferation. FAM134B knockdown increased the protein amount of STIM1, an ER Ca2+ sensor protein mediating the store-operated Ca2+ entry (SOCE) involved in G1 to S phase transition. FAM134B bound to STIM1 through its C-terminal cytosolic region. FAM134B knockdown reduced transport of STIM1 from the ER to autolysosomes. Finally, FAM134B knockdown accelerated G1 to S phase transition. These results suggest that FAM134B is involved in cell proliferation possibly through degradation of STIM1 via ER-phagy.

Keywords:  ER-phagy; FAM134B; STIM1; autophagy; cell cycle; cell proliferation; endoplasmic reticulum (ER); protein degradation

DOI:  https://doi.org/10.1016/j.jbc.2024.107674
Nat Commun. 2024 Aug 09. 15(1): 6810

Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma.

Sinan Xiong, Jianbiao Zhou, Tze King Tan, Tae-Hoon Chung, Tuan Zea Tan, Sabrina Hui-Min Toh, Nicole Xin Ning Tang, Yunlu Jia, Yi Xiang See, Melissa Jane Fullwood, Takaomi Sanda, Wee-Joo Chng.

Multiple myeloma is a hematological malignancy arising from immunoglobulin-secreting plasma cells. It remains poorly understood how chromatin rewiring of regulatory elements contributes to tumorigenesis and therapy resistance in myeloma. Here we generate a high-resolution contact map of myeloma-associated super-enhancers by integrating H3K27ac ChIP-seq and HiChIP from myeloma cell lines, patient-derived myeloma cells and normal plasma cells. Our comprehensive transcriptomic and phenomic analyses prioritize candidate genes with biological and clinical implications in myeloma. We show that myeloma cells frequently acquire SE that transcriptionally activate an oncogene PPP1R15B, which encodes a regulatory subunit of the holophosphatase complex that dephosphorylates translation initiation factor eIF2α. Epigenetic silencing or knockdown of PPP1R15B activates pro-apoptotic eIF2α-ATF4-CHOP pathway, while inhibiting protein synthesis and immunoglobulin production. Pharmacological inhibition of PPP1R15B using Raphin1 potentiates the anti-myeloma effect of bortezomib. Our study reveals that myeloma cells are vulnerable to perturbation of PPP1R15B-dependent protein homeostasis, highlighting a promising therapeutic strategy.

DOI: https://doi.org/10.1038/s41467-024-50910-z
Chem Sci. 2024 Aug 14. 15(32): 12676-12685

Redox-active chemical chaperones exhibiting promiscuous binding promote oxidative protein folding under condensed sub-millimolar conditions.

Koki Suzuki, Ryoya Nojiri, Motonori Matsusaki, Takuya Mabuchi, Shingo Kanemura, Kotone Ishii, Hiroyuki Kumeta, Masaki Okumura, Tomohide Saio, Takahiro Muraoka.

Proteins form native structures through folding processes, many of which proceed through intramolecular hydrophobic effect, hydrogen bond and disulfide-bond formation. In vivo, protein aggregation is prevented even in the highly condensed milieu of a cell through folding mediated by molecular chaperones and oxidative enzymes. Chemical approaches to date have not replicated such exquisite mediation. Oxidoreductases efficiently promote folding by the cooperative effects of oxidative reactivity for disulfide-bond formation in the client unfolded protein and chaperone activity to mitigate aggregation. Conventional synthetic folding promotors mimic the redox-reactivity of thiol/disulfide units but do not address client-recognition units for inhibiting aggregation. Herein, we report thiol/disulfide compounds containing client-recognition units, which act as synthetic oxidoreductase-mimics. For example, compound βCDWSH/SS bears a thiol/disulfide unit at the wide rim of β-cyclodextrin as a client recognition unit. βCDWSH/SS shows promiscuous binding to client proteins, mitigates protein aggregation, and accelerates disulfide-bond formation. In contrast, positioning a thiol/disulfide unit at the narrow rim of β-cyclodextrin promotes folding less effectively through preferential interactions at specific residues, resulting in aggregation. The combination of promiscuous client-binding and redox reactivity is effective for the design of synthetic folding promoters. βCDWSH/SS accelerates oxidative protein folding at highly condensed sub-millimolar protein concentrations.

DOI: https://doi.org/10.1039/d4sc02123a
Nat Commun. 2024 Aug 12. 15(1): 6905

The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function.

Rongrong Zhang, Yu Wu, Xiangru Qu, Wenjuan Yang, Qin Wu, Lin Huang, Qiantao Jiang, Jian Ma, Yazhou Zhang, Pengfei Qi, Guoyue Chen, Yunfeng Jiang, Youliang Zheng, Xiaojie Wang, Yuming Wei, Qiang Xu.

Chloroplasts are key players in photosynthesis and immunity against microbial pathogens. However, the precise and timely regulatory mechanisms governing the control of photosynthesis-associated nuclear genes (PhANGs) expression in plant immunity remain largely unknown. Here we report that TaPIR1, a Pst-induced RING-finger E3 ubiquitin ligase, negatively regulates Pst resistance by specifically interacting with TaHRP1, an atypical transcription factor histidine-rich protein. TaPIR1 ubiquitinates the lysine residues K131 and K136 in TaHRP1 to regulate its stability. TaHRP1 directly binds to the TaHRP1-binding site elements within the PhANGs promoter to activate their transcription via the histidine-rich domain of TaHRP1. PhANGs expression induces the production of chloroplast-derived ROS. Although knocking out TaHRP1 reduces Pst resistance, TaHRP1 overexpression contributes to photosynthesis, and chloroplast-derived ROS production, and improves disease resistance. TaPIR1 expression inhibits the downstream activation of TaHRP1 and TaHRP1-induced ROS accumulation in chloroplasts. Overall, we show that the TaPIR1-mediated ubiquitination and degradation of TaHRP1 alters PhANGs expression to disrupt chloroplast function, thereby increasing plant susceptibility to Pst.

DOI: https://doi.org/10.1038/s41467-024-51249-1
Nat Commun. 2024 Aug 09. 15(1): 6801

Proximity analysis of native proteomes reveals phenotypic modifiers in a mouse model of autism and related neurodevelopmental conditions.

Yudong Gao, Daichi Shonai, Matthew Trn, Jieqing Zhao, Erik J Soderblom, S Alexandra Garcia-Moreno, Charles A Gersbach, William C Wetsel, Geraldine Dawson, Dmitry Velmeshev, Yong-Hui Jiang, Laura G Sloofman, Joseph D Buxbaum, Scott H Soderling.

One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving the identification of high-specificity spatial proteomes. The resulting native proximity proteomes are enriched for human genes dysregulated in the brain of autistic individuals, and reveal proximity interactions between proteins from high-confidence risk genes with those of lower-confidence that may provide new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology.

DOI: https://doi.org/10.1038/s41467-024-51037-x
STAR Protoc. 2024 Aug 09. pii: S2666-1667(24)00418-0. [Epub ahead of print]5(3): 103253

Protocol for the isolation and purification of endoplasmic reticulum-plasma membrane junctions from the mouse brain.

Jason A Weesner, Diantha van de Vlekkert, Leigh Ellen Fremuth, Alessandra d'Azzo.

  Dynamic communication between intracellular organelles often takes place at specialized membrane contact sites that form between their membranes. Here we detail a procedure for the purification of endoplasmic reticulum-plasma membrane (ER-PM) junctions from the mouse brain. We describe steps for homogenizing isolated brain hemispheres and sequential centrifugation to remove the nuclear fraction from the other membrane fractions. We then detail procedures for separating the resulting crude membrane fractions by sucrose density gradients and purifying into their respective pellets. For complete details on the use and execution of this protocol, please refer to Weesner et al.1.

Keywords:  Cell Membrane; Cell separation/fractionation; Neuroscience

DOI:  https://doi.org/10.1016/j.xpro.2024.103253
Nature. 2024 Aug 14.

De novo design of allosterically switchable protein assemblies.

Arvind Pillai, Abbas Idris, Annika Philomin, Connor Weidle, Rebecca Skotheim, Philip J Y Leung, Adam Broerman, Cullen Demakis, Andrew J Borst, Florian Praetorius, David Baker.

Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling1-3. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such 'action at a distance' modulation and to create synthetic proteins whose functions can be regulated by effectors4-7. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge8,9. Here, inspired by the classic Monod-Wyman-Changeux model of cooperativity10, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules11 to protein interfaces12 that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry13 and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin14. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain-side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.

DOI: https://doi.org/10.1038/s41586-024-07813-2
J Cell Biol. 2024 Oct 07. pii: e202304031. [Epub ahead of print]223(10):

Dynamic interaction of REEP5-MFN1/2 enables mitochondrial hitchhiking on tubular ER.

Shue Chen, Yang Sun, Yuling Qin, Lan Yang, Zhenhua Hao, Zhihao Xu, Mikael Björklund, Wei Liu, Zhi Hong.

Mitochondrial functions can be regulated by membrane contact sites with the endoplasmic reticulum (ER). These mitochondria-ER contact sites (MERCs) are functionally heterogeneous and maintained by various tethers. Here, we found that REEP5, an ER tubule-shaping protein, interacts with Mitofusins 1/2 to mediate mitochondrial distribution throughout the cytosol by a new transport mechanism, mitochondrial "hitchhiking" with tubular ER on microtubules. REEP5 depletion led to reduced tethering and increased perinuclear localization of mitochondria. Conversely, increasing REEP5 expression facilitated mitochondrial distribution throughout the cytoplasm. Rapamycin-induced irreversible REEP5-MFN1/2 interaction led to mitochondrial hyperfusion, implying that the dynamic release of mitochondria from tethering is necessary for normal mitochondrial distribution and dynamics. Functionally, disruption of MFN2-REEP5 interaction dynamics by forced dimerization or silencing REEP5 modulated the production of mitochondrial reactive oxygen species (ROS). Overall, our results indicate that dynamic REEP5-MFN1/2 interaction mediates cytosolic distribution and connectivity of the mitochondrial network by "hitchhiking" and this process regulates mitochondrial ROS, which is vital for multiple physiological functions.

DOI: https://doi.org/10.1083/jcb.202304031
Cell Rep. 2024 Aug 08. pii: S2211-1247(24)00937-9. [Epub ahead of print]43(8): 114598

Defective endomembrane dynamics in Rab27a deficiency impairs nucleic acid sensing and cytokine secretion in immune cells.

Juan Yu, Elsa Meneses-Salas, Jennifer L Johnson, Susanna Manenti, Mouad Ait Kbaich, Danni Chen, Kasra Askari, Jing He, Aparna Shukla, Binchu Shaji, Rosana Gonzalez-Quintial, Ben A Croker, Jinzhong Zhang, Hal Hoffman, William B Kiosses, Catherine Hedrick, Kersi Pestonjamasp, Nathan Wineinger, Roberto Baccala, Sergio D Catz.

  Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and β2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo, CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination.

Keywords:  CP: Cell biology; CP: Immunology; CpG; IFN-α; IL-10; Rab GTPase; TLR7; TLR9; amphisome; endosomal Toll-like receptor; neutrophil; plasmacytoid Dendritic Cell

DOI:  https://doi.org/10.1016/j.celrep.2024.114598
Genome Res. 2024 Aug 14. pii: gr.278980.124. [Epub ahead of print]

A spatiotemporally resolved atlas of mRNA decay in the C. elegans embryo reveals differential regulation of mRNA stability across stages and cell types.

Felicia Peng, C Erik Nordgren, John Isaac Murray.

During embryonic development, cells undergo dynamic changes in gene expression that are required for appropriate cell fate specification. Although both transcription and mRNA degradation contribute to gene expression dynamics, patterns of mRNA decay are less well-understood. Here we directly measured spatiotemporally resolved mRNA decay rates transcriptome-wide throughout C. elegans embryogenesis by transcription inhibition followed by bulk and single-cell RNA sequencing. This allowed us to calculate mRNA half-lives within specific cell types and developmental stages and identify differentially regulated mRNA decay throughout embryonic development. We identified transcript features that are correlated with mRNA stability and found that mRNA decay rates are associated with distinct peaks in gene expression over time. Moreover, we provide evidence that, on average, mRNA is more stable in the germline compared to in the soma and in later embryonic stages compared to in earlier stages. This work suggests that differential mRNA decay across cell states and time helps to shape developmental gene expression, and it provides a valuable resource for studies of mRNA turnover regulatory mechanisms.

DOI: https://doi.org/10.1101/gr.278980.124
Nat Commun. 2024 Aug 10. 15(1): 6845

Intestinal Nogo-B reduces GLP1 levels by binding to proglucagon on the endoplasmic reticulum to inhibit PCSK1 cleavage.

Ke Gong, Chao Xue, Zian Feng, Ruru Pan, Mengyao Wang, Shasha Chen, Yuanli Chen, Yudong Guan, Lingyun Dai, Shuang Zhang, Liwei Jiang, Ling Li, Bei Wang, Zequn Yin, Likun Ma, Yasuko Iwakiri, Junming Tang, Chenzhong Liao, Houzao Chen, Yajun Duan.

Glucagon-like peptide 1 (GLP1), which is mainly processed and cleaved from proglucagon in enteroendocrine cells (EECs) of the intestinal tract, acts on the GLP1 receptor in pancreatic cells to stimulate insulin secretion and to inhibit glucagon secretion. However, GLP1 processing is not fully understood. Here, we show that reticulon 4B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, interacts with the major proglucagon fragment of proglucagon to retain proglucagon on the ER, thereby inhibiting PCSK1-mediated cleavage of proglucagon in the Golgi. Intestinal Nogo-B knockout in male type 2 diabetes mellitus (T2DM) mice increases GLP1 and insulin levels and decreases glucagon levels, thereby alleviating pancreatic injury and insulin resistance. Finally, we identify aberrantly elevated Nogo-B expression and inhibited proglucagon cleavage in EECs from diabetic patients. Our study reveals the subcellular regulatory processes involving Nogo-B during GLP1 production and suggests intestinal Nogo-B as a potential therapeutic target for T2DM.

DOI: https://doi.org/10.1038/s41467-024-51352-3
Mol Cell. 2024 Aug 09. pii: S1097-2765(24)00618-X. [Epub ahead of print]

OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response.

Felix G Liang, Fereshteh Zandkarimi, Jaehoon Lee, Joshua L Axelrod, Ryan Pekson, Yisang Yoon, Brent R Stockwell, Richard N Kitsis.

  Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.

Keywords:  ATF4; GPx4; OPA1; cell death; ferroptosis; integrated stress response; mitochondria; system X(c)(−); xCT

DOI:  https://doi.org/10.1016/j.molcel.2024.07.020