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FEBS Letters Oct 2023One of the remarkable features of eukaryotes is the nucleus, delimited by the nuclear envelope (NE), a complex structure and home to the nuclear lamina and nuclear pore... (Review)
Review
One of the remarkable features of eukaryotes is the nucleus, delimited by the nuclear envelope (NE), a complex structure and home to the nuclear lamina and nuclear pore complex (NPC). For decades, these structures were believed to be mainly architectural elements and, in the case of the NPC, simply facilitating nucleocytoplasmic trafficking. More recently, the critical roles of the lamina, NPC and other NE constituents in genome organisation, maintaining chromosomal domains and regulating gene expression have been recognised. Importantly, mutations in genes encoding lamina and NPC components lead to pathogenesis in humans, while pathogenic protozoa disrupt the progression of normal development and expression of pathogenesis-related genes. Here, we review features of the lamina and NPC across eukaryotes and discuss how these elements are structured in trypanosomes, protozoa of high medical and veterinary importance, highlighting lineage-specific and conserved aspects of nuclear organisation.
Topics: Humans; Active Transport, Cell Nucleus; Nuclear Pore Complex Proteins; Nuclear Envelope; Nuclear Pore; Trypanosoma
PubMed: 37789516
DOI: 10.1002/1873-3468.14747 -
Nature Communications Nov 2023Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss...
Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells. These analyses define the UBA1/E2-sensitive proteome and the E2 specificity in protein modulation. Interestingly, profound adaptations in peroxisomes and other organelles are triggered by decreased ubiquitination. While the cargo receptor PEX5 depends on its mono-ubiquitination for binding to peroxisomal proteins and importing them into peroxisomes, we find that UBA1/E2 knockdown induces the compensatory upregulation of other PEX proteins necessary for PEX5 docking to the peroxisomal membrane. Altogether, this study defines a homeostatic mechanism that sustains peroxisomal protein import in cells with decreased ubiquitination capacity.
Topics: Humans; Ubiquitination; Ubiquitin; Protein Transport; Peroxisomes; Intracellular Membranes
PubMed: 37963875
DOI: 10.1038/s41467-023-43262-7 -
EMBO Reports Dec 2023Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61...
Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.
Topics: Animals; SEC Translocation Channels; Membrane Proteins; Endoplasmic Reticulum; Mammals; Peptides; Protein Transport
PubMed: 37983950
DOI: 10.15252/embr.202357910 -
Traffic (Copenhagen, Denmark) Nov 2023Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR...
Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi.
Topics: Toll-Like Receptor 4; Toll-Like Receptor 2; Toll-Like Receptor 3; Toll-Like Receptors; Protein Transport
PubMed: 37491993
DOI: 10.1111/tra.12912 -
Open Biology Aug 2023Encapsulation and compartmentalization are fundamental to the evolution of cellular life, but they also pose a challenge: how to partition the molecules that perform... (Review)
Review
Encapsulation and compartmentalization are fundamental to the evolution of cellular life, but they also pose a challenge: how to partition the molecules that perform biological functions-the proteins-across impermeable barriers into sub-cellular organelles, and to the outside. The solution lies in the evolution of specialized machines, translocons, found in every biological membrane, which act both as gate and gatekeeper across and into membrane bilayers. Understanding how these translocons operate at the molecular level has been a long-standing ambition of cell biology, and one that is approaching its denouement; particularly in the case of the ubiquitous Sec system. In this review, we highlight the fruits of recent game-changing technical innovations in structural biology, biophysics and biochemistry to present a largely complete mechanism for the bacterial version of the core Sec machinery. We discuss the merits of our model over alternative proposals and identify the remaining open questions. The template laid out by the study of the Sec system will be of immense value for probing the many other translocons found in diverse biological membranes, towards the ultimate goal of altering or impeding their functions for pharmaceutical or biotechnological purposes.
Topics: Protein Transport; Cell Membrane
PubMed: 37643640
DOI: 10.1098/rsob.230166 -
Philosophical Transactions of the Royal... Apr 2024The () gene encodes a core component of the retromer complex essential for the endosomal sorting and recycling of transmembrane cargo. Endo-lysosomal pathway deficits... (Review)
Review
The () gene encodes a core component of the retromer complex essential for the endosomal sorting and recycling of transmembrane cargo. Endo-lysosomal pathway deficits are suggested to play a role in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Mutations in cause a late-onset, autosomal dominant form of PD, with a single missense mutation (D620N) shown to segregate with disease in PD families. Understanding how the PD-linked D620N mutation causes retromer dysfunction will provide valuable insight into the pathophysiology of PD and may advance the identification of therapeutics. D620N VPS35 can induce LRRK2 hyperactivation and impair endosomal recruitment of the WASH complex but is also linked to mitochondrial and autophagy-lysosomal pathway dysfunction and altered neurotransmitter receptor transport. The clinical similarities between -linked PD and sporadic PD suggest that defects observed in cellular and animal models with the mutation may provide valuable insights into sporadic disease. In this review, we highlight the current knowledge surrounding VPS35 and its role in retromer dysfunction in PD. We provide a critical discussion of the mechanisms implicated in -mediated neurodegeneration in PD, as well as the interplay between VPS35 and other PD-linked gene products. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.
Topics: Animals; Humans; Parkinson Disease; Vesicular Transport Proteins; Protein Transport; Neurodegenerative Diseases; Mutation
PubMed: 38368930
DOI: 10.1098/rstb.2022.0384 -
The Journal of Cell Biology Feb 2024Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C....
Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C. elegans-polarized intestine epithelia, we recently have revealed that the small GTPase RAB-8/Rab8 serves as an important player in the process. Nonetheless, its function and the relevant UcPS itinerary remain poorly understood. Here, we show that deregulated RAB-8 activity resulted in impaired apical UcPS, which increased sensitivity to infection and environmental stress. We also identified the SNARE VTI-1/Vti1a/b as a new RAB-8-interacting factor involved in the apical UcPS. Besides, RAB-11/Rab11 was capable of recruiting RABI-8/Rabin8 to reduce the guanine nucleotide exchange activity of SMGL-1/GEF toward RAB-8, indicating the necessity of a finely tuned RAB-8/RAB-11 network. Populations of RAB-8- and RAB-11-positive endosomal structures containing the apical UcPS cargo moved toward the apical side. In the absence of RAB-11 or its effectors, the cargo was retained in RAB-8- and RAB-11-positive endosomes, respectively, suggesting that these endosomes are utilized as intermediate carriers for the UcPS.
Topics: Animals; Caenorhabditis elegans; Cell Membrane; Protein Transport; Caenorhabditis elegans Proteins; rab GTP-Binding Proteins; Endosomes
PubMed: 38019180
DOI: 10.1083/jcb.202306107 -
BMC Bioinformatics Nov 2023Determining a protein's quaternary state, i.e. the number of monomers in a functional unit, is a critical step in protein characterization. Many proteins form multimers...
BACKGROUND
Determining a protein's quaternary state, i.e. the number of monomers in a functional unit, is a critical step in protein characterization. Many proteins form multimers for their activity, and over 50% are estimated to naturally form homomultimers. Experimental quaternary state determination can be challenging and require extensive work. To complement these efforts, a number of computational tools have been developed for quaternary state prediction, often utilizing experimentally validated structural information. Recently, dramatic advances have been made in the field of deep learning for predicting protein structure and other characteristics. Protein language models, such as ESM-2, that apply computational natural-language models to proteins successfully capture secondary structure, protein cell localization and other characteristics, from a single sequence. Here we hypothesize that information about the protein quaternary state may be contained within protein sequences as well, allowing us to benefit from these novel approaches in the context of quaternary state prediction.
RESULTS
We generated ESM-2 embeddings for a large dataset of proteins with quaternary state labels from the curated QSbio dataset. We trained a model for quaternary state classification and assessed it on a non-overlapping set of distinct folds (ECOD family level). Our model, named QUEEN (QUaternary state prediction using dEEp learNing), performs worse than approaches that include information from solved crystal structures. However, it successfully learned to distinguish multimers from monomers, and predicts the specific quaternary state with moderate success, better than simple sequence similarity-based annotation transfer. Our results demonstrate that complex, quaternary state related information is included in such embeddings.
CONCLUSIONS
QUEEN is the first to investigate the power of embeddings for the prediction of the quaternary state of proteins. As such, it lays out strengths as well as limitations of a sequence-based protein language model approach, compared to structure-based approaches. Since it does not require any structural information and is fast, we anticipate that it will be of wide use both for in-depth investigation of specific systems, as well as for studies of large sets of protein sequences. A simple colab implementation is available at: https://colab.
RESEARCH
google.com/github/Furman-Lab/QUEEN/blob/main/QUEEN_prediction_notebook.ipynb .
Topics: Proteins; Amino Acid Sequence; Language; Protein Structure, Secondary; Protein Transport
PubMed: 37964216
DOI: 10.1186/s12859-023-05549-w -
The Journal of Cell Biology Jul 2023ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possibly redundant functions of the...
ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possibly redundant functions of the five human ARFs make investigating their function a challenging task. To shed light on the roles of the different Golgi-localized ARF members in membrane trafficking, we generated CRISPR-Cas9 knockins (KIs) of type I (ARF1 and ARF3) and type II ARFs (ARF4 and ARF5) and mapped their nanoscale localization with stimulated emission depletion (STED) super-resolution microscopy. We find ARF1, ARF4, and ARF5 on segregated nanodomains on the cis-Golgi and ER-Golgi intermediate compartments (ERGIC), revealing distinct roles in COPI recruitment on early secretory membranes. Interestingly, ARF4 and ARF5 define Golgi-tethered ERGIC elements decorated by COPI and devoid of ARF1. Differential localization of ARF1 and ARF4 on peripheral ERGICs suggests the presence of functionally different classes of intermediate compartments that could regulate bi-directional transport between the ER and the Golgi. Furthermore, ARF1 and ARF3 localize to segregated nanodomains on the trans-Golgi network (TGN) and are found on TGN-derived post-Golgi tubules, strengthening the idea of distinct roles in post-Golgi sorting. This work provides the first map of the nanoscale organization of human ARF GTPases on cellular membranes and sets the stage to dissect their numerous cellular roles.
Topics: Humans; ADP-Ribosylation Factors; Golgi Apparatus; trans-Golgi Network; Protein Transport; Biological Transport; ADP-Ribosylation Factor 1
PubMed: 37102998
DOI: 10.1083/jcb.202205107 -
PLoS Pathogens Jul 2023In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral...
In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.
Topics: Humans; Active Transport, Cell Nucleus; Endothelial Cells; Influenza A virus; Influenza A Virus, H1N1 Subtype; Karyopherins; Kelch-Like ECH-Associated Protein 1; NF-E2-Related Factor 2; Ribonucleoproteins; RNA, Messenger; Virus Replication
PubMed: 37459366
DOI: 10.1371/journal.ppat.1011506