-
Cell Nov 2018Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture...
Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
Topics: Animals; Chromatin; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Drosophila; Gene Knockout Techniques; HEK293 Cells; Humans; Mass Spectrometry; Mutagenesis; Protein Subunits; Recombinant Proteins; Transcription Factors
PubMed: 30343899
DOI: 10.1016/j.cell.2018.09.032 -
Neuropharmacology Oct 2020Nicotine is a highly addictive drug found in tobacco that drives its continued use despite the harmful consequences. The initiation of nicotine abuse involves the... (Review)
Review
Nicotine is a highly addictive drug found in tobacco that drives its continued use despite the harmful consequences. The initiation of nicotine abuse involves the mesolimbic dopamine system, which contributes to the rewarding sensory stimuli and associative learning processes in the beginning stages of addiction. Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs), which come in a diverse collection of subtypes. The nAChRs that contain the α4 and β2 subunits, often in combination with the α6 subunit, are particularly important for nicotine's ability to increase midbrain dopamine neuron firing rates and phasic burst firing. Chronic nicotine exposure results in numerous neuroadaptations, including the upregulation of particular nAChR subtypes associated with long-term desensitization of the receptors. When nicotine is no longer present, for example during attempts to quit smoking, a withdrawal syndrome develops. The expression of physical withdrawal symptoms depends mainly on the α2, α3, α5, and β4 nicotinic subunits in the epithalamic habenular complex and its target regions. Thus, nicotine affects diverse neural systems and an array of nAChR subtypes to mediate the overall addiction process. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.
Topics: Animals; Brain; Humans; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Protein Subunits; Receptors, Nicotinic; Tobacco Use Disorder
PubMed: 32738308
DOI: 10.1016/j.neuropharm.2020.108256 -
Nature Oct 2016Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the...
Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.
Topics: Cell Line; Cell Respiration; Cell Survival; Electron Transport Complex I; Gene Editing; Gene Knockout Techniques; HEK293 Cells; Humans; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Mitochondrial Proton-Translocating ATPases; Models, Molecular; Protein Stability; Protein Subunits; Proteomics
PubMed: 27626371
DOI: 10.1038/nature19754 -
Cellular and Molecular Life Sciences :... Dec 2014In eukaryotic cells, proteasomes are highly conserved protease complexes and eliminate unwanted proteins which are marked by poly-ubiquitin chains for degradation. The... (Review)
Review
In eukaryotic cells, proteasomes are highly conserved protease complexes and eliminate unwanted proteins which are marked by poly-ubiquitin chains for degradation. The 26S proteasome consists of the proteolytic core particle, the 20S proteasome, and the 19S regulatory particle, which are composed of 14 and 19 different subunits, respectively. Proteasomes are the second-most abundant protein complexes and are continuously assembled from inactive precursor complexes in proliferating cells. The modular concept of proteasome assembly was recognized in prokaryotic ancestors and applies to eukaryotic successors. The efficiency and fidelity of eukaryotic proteasome assembly is achieved by several proteasome-dedicated chaperones that initiate subunit incorporation and control the quality of proteasome assemblies by transiently interacting with proteasome precursors. It is important to understand the mechanism of proteasome assembly as the proteasome has key functions in the turnover of short-lived proteins regulating diverse biological processes.
Topics: Animals; Humans; Models, Biological; Models, Molecular; Proteasome Endopeptidase Complex; Protein Binding; Protein Conformation; Protein Subunits
PubMed: 25107634
DOI: 10.1007/s00018-014-1699-8 -
Frontiers in Immunology 2023Tuberculosis (TB), also known as the "White Plague", is caused by (). Before the COVID-19 epidemic, TB had the highest mortality rate of any single infectious disease.... (Review)
Review
Tuberculosis (TB), also known as the "White Plague", is caused by (). Before the COVID-19 epidemic, TB had the highest mortality rate of any single infectious disease. Vaccination is considered one of the most effective strategies for controlling TB. Despite the limitations of the Bacille Calmette-Guérin (BCG) vaccine in terms of protection against TB among adults, it is currently the only licensed TB vaccine. Recently, with the evolution of bioinformatics and structural biology techniques to screen and optimize protective antigens of , the tremendous potential of protein subunit vaccines is being exploited. Multistage subunit vaccines obtained by fusing immunodominant antigens from different stages of TB infection are being used both to prevent and to treat TB. Additionally, the development of novel adjuvants is compensating for weaknesses of immunogenicity, which is conducive to the flourishing of subunit vaccines. With advances in the development of animal models, preclinical vaccine protection assessments are becoming increasingly accurate. This review summarizes progress in the research of protein subunit TB vaccines during the past decades to facilitate the further optimization of protein subunit vaccines that may eradicate TB.
Topics: Animals; Protein Subunits; COVID-19; Vaccines, Subunit; Tuberculosis; Tuberculosis Vaccines; BCG Vaccine
PubMed: 37654500
DOI: 10.3389/fimmu.2023.1238586 -
Methods in Molecular Biology (Clifton,... 2021Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of...
Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of membrane proteins and their complexes is of key importance, but the progress in this area is slow because of the difficulties to produce them in sufficient quality and quantity. Overexpression of membrane proteins is often restricted by the limited capability of translocation systems to integrate proteins into the membrane and to fold them properly. Purification of membrane proteins requires their isolation from the membrane, which is a further challenge. The choice of expression system, detergents, and purification tags is therefore an important decision. Here, we present a protocol for expression in bacteria and isolation of a seven-subunit membrane protein complex, the bacterial holo-translocon, which can serve as a starting point for the production of other membrane protein complexes for structural and functional studies.
Topics: Chromatography, Affinity; Chromatography, Gel; Escherichia coli; Gene Expression; Membrane Proteins; Multiprotein Complexes; Plasmids; Promoter Regions, Genetic; Protein Multimerization; Protein Subunits; Recombinant Proteins
PubMed: 33301109
DOI: 10.1007/978-1-0716-1126-5_1 -
Frontiers in Bioscience (Landmark... Jan 2017V-ATPases are ATP-driven proton pumps present in both intracellular and cell surface membranes of eukaryotes that function in many normal and disease processes.... (Review)
Review
V-ATPases are ATP-driven proton pumps present in both intracellular and cell surface membranes of eukaryotes that function in many normal and disease processes. V-ATPases are large, multi-subunit complexes composed of a peripheral domain (V) that hydrolyzes ATP and a membrane integral domain (V) that translocates protons. Because of the diversity of their functions, V-ATPase activity is controlled by a number of mechanisms. Regulated assembly of the V and V domains rapidly modulates V-ATPase activity in response to a variety of cues, including nutrient availability, growth factor stimulation and cellular differentiation. Considerable information has recently emerged concerning the cellular signaling pathways controlling regulated assembly. Acid secretion by epithelial cells in the kidney and epididymus is controlled by regulated trafficking of V-ATPases to the cell surface. Isoforms of subunit a of the V domain both control trafficking of V-ATPases to distinct cellular membranes and confer properties to the resultant complexes that help account for differences in pH between cellular compartments. Finally, differential expression of genes encoding V-ATPases subunits occurs in a number of contexts, including cancer.
Topics: Animals; Humans; Insect Proteins; Mammals; Models, Molecular; Protein Multimerization; Protein Subunits; Protein Transport; Saccharomyces cerevisiae Proteins; Vacuolar Proton-Translocating ATPases
PubMed: 27814636
DOI: 10.2741/4506 -
Trends in Biochemical Sciences Jun 2016The target of rapamycin (TOR) kinase functions in two multiprotein complexes, TORC1 and TORC2. Although both complexes are evolutionarily conserved, only TORC1 is... (Review)
Review
The target of rapamycin (TOR) kinase functions in two multiprotein complexes, TORC1 and TORC2. Although both complexes are evolutionarily conserved, only TORC1 is acutely inhibited by rapamycin. Consequently, only TORC1 signaling is relatively well understood; and, at present, only mammalian TORC1 is a validated drug target, pursued in immunosuppression and oncology. However, the knowledge void surrounding TORC2 is dissipating. Acute inhibition of TORC2 with small molecules is now possible and structural studies of both TORC1 and TORC2 have recently been reported. Here we review these recent advances as well as observations made from tissue-specific mTORC2 knockout mice. Together these studies help define TORC2 structure-function relationships and suggest that mammalian TORC2 may one day also become a bona fide clinical target.
Topics: Animals; Antibiotics, Antineoplastic; Binding Sites; Gene Expression Regulation; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Knockout; Models, Molecular; Multiprotein Complexes; Protein Binding; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Protein Subunits; Signal Transduction; Sirolimus; Structure-Activity Relationship; TOR Serine-Threonine Kinases
PubMed: 27161823
DOI: 10.1016/j.tibs.2016.04.001 -
Current Opinion in Structural Biology Oct 2016With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments... (Review)
Review
With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes. Importantly we also show how developments in mass spectrometry can define optimal solution conditions to guide downstream structure determination, particularly of challenging biomolecules that refuse to crystallise.
Topics: Biology; Crystallography; DNA; Humans; Mass Spectrometry; Protein Subunits; RNA
PubMed: 27721169
DOI: 10.1016/j.sbi.2016.09.008 -
Bioorganic Chemistry Mar 2023The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for...
The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for selective labeling of biomolecules. In this work, we exploit the slow reaction between a non-conjugated terminal alkene and a tetrazine, and apply this reaction to achieving a proximity-enhanced protein crosslinking. In one protein subunit, a terminal alkene-containing amino acid was site-specifically incorporated in response to an amber nonsense codon. In another protein subunit, a tetrazine moiety was introduced through the attachment to a cysteine residue. Fast protein crosslinking was achieved due to a large increase in effective molarity of the two reactants that were brought to close proximity by the two interacting protein subunits. Such a proximity-enhanced protein crosslinking is useful for the study of protein-protein interactions.
Topics: Alkenes; Protein Subunits; Heterocyclic Compounds; Amino Acids; Cycloaddition Reaction
PubMed: 36642019
DOI: 10.1016/j.bioorg.2023.106359