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Molecular Cell Aug 2023Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate...
Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.
Topics: Humans; Acclimatization; Mitochondria; Saccharomyces cerevisiae; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Mitochondrial Proteins
PubMed: 37595558
DOI: 10.1016/j.molcel.2023.07.021 -
Nature Sep 2023Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical...
Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.
Topics: Cryoelectron Microscopy; Mitochondrial Precursor Protein Import Complex Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Mitochondria
PubMed: 37344598
DOI: 10.1038/s41586-023-06239-6 -
Molecular Cell Oct 2023p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal...
p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.
Topics: Mice; Rats; Humans; Animals; Autophagosomes; Ubiquitinated Proteins; Sequestosome-1 Protein; Autophagy; Acylation; Microtubule-Associated Proteins; Mammals
PubMed: 37802024
DOI: 10.1016/j.molcel.2023.09.004 -
Nature Aug 2023Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation. The Rpd3 small (Rpd3S) complex recognizes histone H3...
Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3-Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex. Multivalent recognition of two H3K36me3 marks, nucleosomal DNA and linker DNAs by Eaf3, Sin3 and Rco1 positions the catalytic centre of Rpd3 next to the histone H4 N-terminal tail for deacetylation. In an alternative catalytic mode, combinatorial readout of unmethylated histone H3 lysine 4 and H3K36me3 by Rco1 and Eaf3 directs histone H3-specific deacetylation except for the registered histone H3 acetylated lysine 9. Collectively, our work illustrates dynamic and diverse modes of multivalent nucleosomal engagement and methylation-guided deacetylation by Rpd3S, highlighting the exquisite complexity of epigenetic regulation with delicately designed multi-subunit enzymatic machineries in transcription and beyond.
Topics: Acetylation; Cryoelectron Microscopy; DNA, Fungal; Epigenesis, Genetic; Histones; Lysine; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Methylation; Multiprotein Complexes
PubMed: 37468628
DOI: 10.1038/s41586-023-06349-1 -
Nature Jan 2024AlphaFold2 (ref. ) has revolutionized structural biology by accurately predicting single structures of proteins. However, a protein's biological function often depends...
AlphaFold2 (ref. ) has revolutionized structural biology by accurately predicting single structures of proteins. However, a protein's biological function often depends on multiple conformational substates, and disease-causing point mutations often cause population changes within these substates. We demonstrate that clustering a multiple-sequence alignment by sequence similarity enables AlphaFold2 to sample alternative states of known metamorphic proteins with high confidence. Using this method, named AF-Cluster, we investigated the evolutionary distribution of predicted structures for the metamorphic protein KaiB and found that predictions of both conformations were distributed in clusters across the KaiB family. We used nuclear magnetic resonance spectroscopy to confirm an AF-Cluster prediction: a cyanobacteria KaiB variant is stabilized in the opposite state compared with the more widely studied variant. To test AF-Cluster's sensitivity to point mutations, we designed and experimentally verified a set of three mutations predicted to flip KaiB from Rhodobacter sphaeroides from the ground to the fold-switched state. Finally, screening for alternative states in protein families without known fold switching identified a putative alternative state for the oxidoreductase Mpt53 in Mycobacterium tuberculosis. Further development of such bioinformatic methods in tandem with experiments will probably have a considerable impact on predicting protein energy landscapes, essential for illuminating biological function.
Topics: Cluster Analysis; Mutation; Protein Conformation; Proteins; Sequence Alignment; Machine Learning; Rhodobacter sphaeroides; Bacterial Proteins; Protein Folding
PubMed: 37956700
DOI: 10.1038/s41586-023-06832-9 -
Nature Reviews. Molecular Cell Biology Jun 2024Over the past two decades, protein S-acylation (often referred to as S-palmitoylation) has emerged as an important regulator of vital signalling pathways. S-Acylation is... (Review)
Review
Over the past two decades, protein S-acylation (often referred to as S-palmitoylation) has emerged as an important regulator of vital signalling pathways. S-Acylation is a reversible post-translational modification that involves the attachment of a fatty acid to a protein. Maintenance of the equilibrium between protein S-acylation and deacylation has demonstrated profound effects on various cellular processes, including innate immunity, inflammation, glucose metabolism and fat metabolism, as well as on brain and heart function. This Review provides an overview of current understanding of S-acylation and deacylation enzymes, their spatiotemporal regulation by sophisticated multilayered mechanisms, and their influence on protein function, cellular processes and physiological pathways. Furthermore, we examine how disruptions in protein S-acylation are associated with a broad spectrum of diseases from cancer to autoinflammatory disorders and neurological conditions.
Topics: Humans; Animals; Acylation; Protein Processing, Post-Translational; Signal Transduction; Lipoylation; Proteins
PubMed: 38355760
DOI: 10.1038/s41580-024-00700-8 -
Cell Reports Mar 2024Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hinders host gene expression, curbing defenses and licensing viral protein synthesis and virulence. During...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hinders host gene expression, curbing defenses and licensing viral protein synthesis and virulence. During SARS-CoV-2 infection, the virulence factor non-structural protein 1 (Nsp1) targets the mRNA entry channel of mature cytoplasmic ribosomes, limiting translation. We show that Nsp1 also restrains translation by targeting nucleolar ribosome biogenesis. SARS-CoV-2 infection disrupts 18S and 28S ribosomal RNA (rRNA) processing. Expression of Nsp1 recapitulates the processing defects. Nsp1 abrogates rRNA production without altering the expression of critical processing factors or nucleolar organization. Instead, Nsp1 localizes to the nucleolus, interacting with precursor-rRNA and hindering its maturation separately from the viral protein's role in restricting mature ribosomes. Thus, SARS-CoV-2 Nsp1 limits translation by targeting ribosome biogenesis and mature ribosomes. These findings revise our understanding of how SARS-CoV-2 Nsp1 controls human protein synthesis, suggesting that efforts to counter Nsp1's effect on translation should consider the protein's impact from ribosome manufacturing to mature ribosomes.
Topics: Humans; SARS-CoV-2; RNA, Ribosomal; COVID-19; Ribosomes; Viral Proteins; Viral Nonstructural Proteins
PubMed: 38427561
DOI: 10.1016/j.celrep.2024.113891 -
Progress in Molecular Biology and... 2024Protein aggregation is a complex process with several stages that lead to the formation of complex structures and shapes with a broad variability in stability and... (Review)
Review
Protein aggregation is a complex process with several stages that lead to the formation of complex structures and shapes with a broad variability in stability and toxicity. The aggregation process is affected by various factors and environmental conditions that disrupt the protein's original state, including internal factors like mutations, expression levels, and polypeptide chain truncation, as well as external factors, such as dense molecular surroundings, post-translation modifications, and interactions with other proteins, nucleic acids, small molecules, metal ions, chaperones, and lipid membranes. During the aggregation process, the biological activity of an aggregating protein may be reduced or eliminated, whereas the resulting aggregates may have the potential to be immunogenic, or they may have other undesirable properties. Finding the cause(s) of protein aggregation and controlling it to an acceptable level is among the most crucial topics of research in academia and biopharmaceutical companies. This chapter aims to review intrinsic pathways of protein aggregation and potential extrinsic variables that influence this process.
Topics: Amyloid; Humans; Animals; Protein Aggregates
PubMed: 38811089
DOI: 10.1016/bs.pmbts.2024.03.015 -
Trends in Biochemical Sciences Sep 2023The plethora of biological functions that sustain life is rooted in the remarkable evolvability of proteins. An emerging view highlights the importance of a protein's... (Review)
Review
The plethora of biological functions that sustain life is rooted in the remarkable evolvability of proteins. An emerging view highlights the importance of a protein's initial state in dictating evolutionary success. A deeper comprehension of the mechanisms that govern the evolvability of these initial states can provide invaluable insights into protein evolution. In this review, we describe several molecular determinants of protein evolvability, unveiled by experimental evolution and ancestral sequence reconstruction studies. We further discuss how genetic variation and epistasis can promote or constrain functional innovation and suggest putative underlying mechanisms. By establishing a clear framework for these determinants, we provide potential indicators enabling the forecast of suitable evolutionary starting points and delineate molecular mechanisms in need of deeper exploration.
Topics: Proteins; Evolution, Molecular; Biological Evolution
PubMed: 37330341
DOI: 10.1016/j.tibs.2023.05.009 -
The Journal of Biological Chemistry Aug 2023The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes...
The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.
Topics: Anti-Infective Agents; Drug Resistance, Fungal; Endoplasmic Reticulum-Associated Degradation; Hygromycin B; Lipids; Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37331602
DOI: 10.1016/j.jbc.2023.104939