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The EMBO Journal Aug 2023UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT domain-containing ubiquitin ligase has recently been...
UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT domain-containing ubiquitin ligase has recently been identified as an important regulator of oncogenes, e.g., MYC, but little is known about its structure or mechanisms of substrate engagement and ubiquitination. Here, we present the cryo-EM structure of human UBR5, revealing an α-solenoid scaffold with numerous protein-protein interacting motifs, assembled into an antiparallel dimer that adopts further oligomeric states. Using cryo-EM processing tools, we observe the dynamic nature of the UBR5 catalytic domain, which we postulate is important for its enzymatic activity. We characterise the proteasomal nuclear import factor AKIRIN2 as an interacting protein and propose UBR5 as an efficient ubiquitin chain elongator. This preference for ubiquitinated substrates and several distinct domains for protein-protein interactions may explain how UBR5 is linked to several different signalling pathways and cancers. Together, our data expand on the limited knowledge of the structure and function of HECT E3 ligases.
Topics: Humans; Ubiquitin-Protein Ligases; Cryoelectron Microscopy; Ubiquitination; Amino Acid Motifs; Ubiquitin
PubMed: 37409633
DOI: 10.15252/embj.2022113348 -
Molecular Neurodegeneration Jan 2024Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders on a disease spectrum that are characterized by the... (Review)
Review
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders on a disease spectrum that are characterized by the cytoplasmic mislocalization and aberrant phase transitions of prion-like RNA-binding proteins (RBPs). The common accumulation of TAR DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), and other nuclear RBPs in detergent-insoluble aggregates in the cytoplasm of degenerating neurons in ALS/FTD is connected to nuclear pore dysfunction and other defects in the nucleocytoplasmic transport machinery. Recent advances suggest that beyond their canonical role in the nuclear import of protein cargoes, nuclear-import receptors (NIRs) can prevent and reverse aberrant phase transitions of TDP-43, FUS, and related prion-like RBPs and restore their nuclear localization and function. Here, we showcase the NIR family and how they recognize cargo, drive nuclear import, and chaperone prion-like RBPs linked to ALS/FTD. We also discuss the promise of enhancing NIR levels and developing potentiated NIR variants as therapeutic strategies for ALS/FTD and related neurodegenerative proteinopathies.
Topics: Humans; Frontotemporal Dementia; Amyotrophic Lateral Sclerosis; Active Transport, Cell Nucleus; DNA-Binding Proteins; Prions
PubMed: 38254150
DOI: 10.1186/s13024-023-00698-1 -
The Journal of Cell Biology Oct 2023The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70....
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.
Topics: Humans; Coronavirus Nucleocapsid Proteins; COVID-19; HEK293 Cells; Membrane Proteins; Mitochondrial Membrane Transport Proteins; Saccharomyces cerevisiae; SARS-CoV-2
PubMed: 37682539
DOI: 10.1083/jcb.202303002 -
Cellular & Molecular Biology Letters Jan 2024Septic cardiomyopathy (SCM), a common cardiovascular comorbidity of sepsis, has emerged among the leading causes of death in patients with sepsis. SCM's pathogenesis is...
New insights into the role of mitochondrial metabolic dysregulation and immune infiltration in septic cardiomyopathy by integrated bioinformatics analysis and experimental validation.
BACKGROUND
Septic cardiomyopathy (SCM), a common cardiovascular comorbidity of sepsis, has emerged among the leading causes of death in patients with sepsis. SCM's pathogenesis is strongly affected by mitochondrial metabolic dysregulation and immune infiltration disorder. However, the specific mechanisms and their intricate interactions in SCM remain unclear. This study employed bioinformatics analysis and drug discovery approaches to identify the regulatory molecules, distinct functions, and underlying interactions of mitochondrial metabolism and immune microenvironment, along with potential interventional strategies in SCM.
METHODS
GSE79962, GSE171546, and GSE167363 datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and module genes were identified using Limma and Weighted Correlation Network Analysis (WGCNA), followed by functional enrichment analysis. Machine learning algorithms, including support vector machine-recursive feature elimination (SVM-RFE), least absolute shrinkage and selection operator (LASSO) regression, and random forest, were used to screen mitochondria-related hub genes for early diagnosis of SCM. Subsequently, a nomogram was developed based on six hub genes. The immunological landscape was evaluated by single-sample gene set enrichment analysis (ssGSEA). We also explored the expression pattern of hub genes and distribution of mitochondria/inflammation-related pathways in UMAP plots of single-cell dataset. Potential drugs were explored using the Drug Signatures Database (DSigDB). In vivo and in vitro experiments were performed to validate the pathogenetic mechanism of SCM and the therapeutic efficacy of candidate drugs.
RESULTS
Six hub mitochondria-related DEGs [MitoDEGs; translocase of inner mitochondrial membrane domain-containing 1 (TIMMDC1), mitochondrial ribosomal protein S31 (MRPS31), F-box only protein 7 (FBXO7), phosphatidylglycerophosphate synthase 1 (PGS1), LYR motif containing 7 (LYRM7), and mitochondrial chaperone BCS1 (BCS1L)] were identified. The diagnostic nomogram model based on the six hub genes demonstrated high reliability and validity in both the training and validation sets. The immunological microenvironment differed between SCM and control groups. The Spearman correlation analysis revealed that hub MitoDEGs were significantly associated with the infiltration of immune cells. Upregulated hub genes showed remarkably high expression in the naive/memory B cell, CD14 monocyte, and plasma cell subgroup, evidenced by the feature plot. The distribution of mitochondria/inflammation-related pathways varied across subgroups among control and SCM individuals. Metformin was predicted to be the most promising drug with the highest combined score. Its efficacy in restoring mitochondrial function and suppressing inflammatory responses has also been validated.
CONCLUSIONS
This study presents a comprehensive mitochondrial metabolism and immune infiltration landscape in SCM, providing a potential novel direction for the pathogenesis and medical intervention of SCM.
Topics: Humans; Reproducibility of Results; Mitochondria; Cardiomyopathies; DNA, Mitochondrial; Computational Biology; Inflammation; Sepsis; Mitochondrial Precursor Protein Import Complex Proteins; ATPases Associated with Diverse Cellular Activities; Electron Transport Complex III; Molecular Chaperones; Mitochondrial Proteins
PubMed: 38291374
DOI: 10.1186/s11658-024-00536-2 -
Blood Jul 2023
Topics: Humans; Leukemia, Large Granular Lymphocytic; Leukemia, Prolymphocytic, T-Cell; Cell Proliferation; Transferrins
PubMed: 37498584
DOI: 10.1182/blood.2023020479 -
Biological Chemistry Jul 2023Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an...
Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes - TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.
Topics: Protein Transport; Saccharomyces cerevisiae; Mitochondria; Mitochondrial Proteins; Mitochondrial Precursor Protein Import Complex Proteins
PubMed: 37155927
DOI: 10.1515/hsz-2023-0133 -
IUBMB Life Oct 2023The abundance of each cellular protein is dynamically adjusted to the prevailing metabolic and stress conditions by modulation of their synthesis and degradation rates.... (Review)
Review
The abundance of each cellular protein is dynamically adjusted to the prevailing metabolic and stress conditions by modulation of their synthesis and degradation rates. The proteasome represents the major machinery for the degradation of proteins in eukaryotic cells. How the ubiquitin-proteasome system (UPS) controls protein levels and removes superfluous and damaged proteins from the cytosol and the nucleus is well characterized. However, recent studies showed that the proteasome also plays a crucial role in mitochondrial protein quality control. This mitochondria-associated degradation (MAD) thereby acts on two layers: first, the proteasome removes mature, functionally compromised or mis-localized proteins from the mitochondrial surface; and second, the proteasome cleanses the mitochondrial import pore of import intermediates of nascent proteins that are stalled during translocation. In this review, we provide an overview about the components and their specific functions that facilitate proteasomal degradation of mitochondrial proteins in the yeast Saccharomyces cerevisiae. Thereby we explain how the proteasome, in conjunction with a set of intramitochondrial proteases, maintains mitochondrial protein homeostasis and dynamically adapts the levels of mitochondrial proteins to specific conditions.
Topics: Proteasome Endopeptidase Complex; Mitochondrial Proteins; Mitochondria; Ubiquitin; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37178401
DOI: 10.1002/iub.2734 -
Cell Reports Mar 2024Resting CD4 T cells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to...
Resting CD4 T cells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to infection, presumably by alleviating blocks at cytoplasmic reverse transcription and subsequent nuclear import of reverse-transcription/pre-integration complexes (RTC/PICs). Here, spatial analyses using quantitative virus imaging techniques reveal that HIV-1 capsids containing RTC/PICs are readily imported into the nucleus, recruit the host dependency factor CPSF6, and translocate to nuclear speckles in resting CD4 T cells. Reverse transcription, however, remains incomplete, impeding proviral integration and viral gene expression. Vpx or pharmacological inhibition of the deoxynucleotide triphosphohydrolase (dNTPase) activity of the restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) increases levels of nuclear reverse-transcribed cDNA and facilitates HIV-1 integration. Nuclear import and intranuclear transport of viral complexes therefore do not pose important blocks to HIV-1 in resting CD4 T cells, and the limitation to reverse transcription by SAMHD1's dNTPase activity constitutes the main pre-integration block to infection.
Topics: Animals; Humans; HIV-1; CD4-Positive T-Lymphocytes; SAM Domain and HD Domain-Containing Protein 1; HIV Infections; HIV-2; HIV Seropositivity; Viral Regulatory and Accessory Proteins; Monomeric GTP-Binding Proteins; HEK293 Cells
PubMed: 38478523
DOI: 10.1016/j.celrep.2024.113941 -
Haematologica Aug 2023RNA-binding proteins (RBP) have emerged as essential regulators that control gene expression and modulate multiple cancer traits. T-cell acute lymphoblastic leukemia...
RNA-binding proteins (RBP) have emerged as essential regulators that control gene expression and modulate multiple cancer traits. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from transformation of T-cell progenitors that normally undergo discrete steps of differentiation in the thymus. The implications of essential RBP during T-cell neoplastic transformation remain largely unclear. Systematic evaluation of RBP identifies RNA helicase DHX15, which facilitates the disassembly of the spliceosome and release of lariat introns, as a T-ALL dependency factor. Functional analysis using multiple murine T-ALL models demonstrates the essential importance of DHX15 in tumor cell survival and leukemogenesis. Moreover, single-cell transcriptomics reveals that DHX15 depletion in T-cell progenitors hinders burst proliferation during the transition from doublenegative to double-positive cells (CD4-CD8- to CD4+CD8+). Mechanistically, abrogation of DHX15 perturbs RNA splicing and leads to diminished levels of SLC7A6 and SLC38A5 transcripts due to intron retention, thereby suppressing glutamine import and mTORC1 activity. We further propose a DHX15 signature modulator drug ciclopirox and demonstrate that it has prominent anti-T-ALL efficacy. Collectively, our data highlight the functional contribution of DHX15 to leukemogenesis through regulation of established oncogenic pathways. These findings also suggest a promising therapeutic approach, i.e., splicing perturbation by targeting spliceosome disassembly, may achieve considerable anti-tumor efficacy.
Topics: Humans; Animals; Mice; RNA Helicases; RNA Splicing; Spliceosomes; Leukemia; Amino Acid Transport Systems, Basic
PubMed: 36861414
DOI: 10.3324/haematol.2022.282066 -
Nature Sep 2023The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria. About...
The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria. About 60% of more than 1,000 different mitochondrial proteins are synthesized with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices. TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views, including regulatory and coupling functions, have been reported on the essential TIM23 subunit Tim17 (refs. ). Here we mapped the interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins during translocation in the native membrane environment. We show that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane.
Topics: Mitochondria; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37527780
DOI: 10.1038/s41586-023-06477-8