RESUMEN
Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.
Asunto(s)
Saltamontes , Toxinas Biológicas , Sistemas de Secreción Tipo VII , Animales , Sistemas de Secreción Tipo VII/genética , CitoplasmaRESUMEN
Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.
Asunto(s)
Citoesqueleto , Septinas , Actinas , Animales , Citoesqueleto/metabolismo , Humanos , Mamíferos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Septinas/genética , Septinas/metabolismoRESUMEN
Transmissible spongiform encephalopathies (TSEs) or prion diseases consist of a broad range of fatal neurological disorders affecting humans and animals. Contrary to Watson and Crick's 'central dogma', prion diseases are caused by a protein, devoid of DNA involvement. Herein, we briefly review various cellular and biological aspects of prions and prion pathogenesis focusing mainly on historical milestones, biosynthesis, degradation, structure-function of cellular and scrapie forms of prions .
Asunto(s)
Enfermedades por Prión , Priones , Scrapie , Animales , Ovinos , Humanos , Scrapie/genética , Scrapie/metabolismo , Scrapie/patología , Priones/genética , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patologíaRESUMEN
Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this ß7-ß8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by ß7-ß8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Dominio Catalítico , Cisteína Endopeptidasas/química , Mutación , Ingeniería de Proteínas/métodos , Infecciones Estafilocócicas/enzimología , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Staphylococcus aureus/química , Staphylococcus aureus/aislamiento & purificación , Especificidad por SustratoRESUMEN
Tau can adopt distinct fibril conformations in different human neurodegenerative diseases, which may invoke distinct pathological mechanisms. In a recent issue, Weismiller et al. showed that intramolecular disulfide links between cys291 and cys322 for a specific tau isoform containing four microtubule-binding repeats direct the formation of a structurally distinct amyloid polymorph. These findings have implications in how oxidative stress can flip switches of tau polymorphism in these diseases.
Asunto(s)
Amiloidosis , Cisteína , Amiloide/genética , Proteínas Amiloidogénicas , Humanos , Proteínas tau/genéticaRESUMEN
Chemical glycosylation of proteins is a powerful tool applied widely in biomedicine and biotechnology. However, it is a challenging undertaking and typically relies on recombinant proteins and site-specific conjugations. The scope and utility of this nature-inspired methodology would be broadened tremendously by the advent of facile, scalable techniques in glycosylation, which are currently missing. In this work, we investigated a one-pot aqueous protocol to achieve indiscriminate, surface-wide glycosylation of the surface accessible amines (lysines and/or N-terminus). We reveal that this approach afforded minimal if any change in the protein activity and recognition events in biochemical and cell culture assays, but at the same time provided a significant benefit of stabilizing proteins against aggregation and fibrillation - as demonstrated on serum proteins (albumins and immunoglobulin G, IgG), an enzyme (uricase), and proteins involved in neurodegenerative disease (α-synuclein) and diabetes (insulin). Most importantly, this highly advantageous result was achieved via a one-pot aqueous protocol performed on native proteins, bypassing the use of complex chemical methodologies and recombinant proteins.
Asunto(s)
Enfermedades Neurodegenerativas , Glicosilación , LisinaRESUMEN
Malaria did not die with the end of the age of western colonization but is still a major public health issue in large parts of the world. Despite repeated and concerted efforts to eradicate this disease, it has proved remarkably resilient, and constant vigilance and continuous research are required to discover new chinks in the parasite's armor and alleviate the suffering at both the individual and societal levels. A deeper understanding of the fundamental processes underlying parasite survival, propagation, virulence, and ability to cause disease is the key to the development of desperately needed new therapies and prophylactic drugs. Malaria parasites, by the nature of their lifecycle, are subject to a number of environmental and cellular stresses which they must overcome to survive. To this end, they express a number of heat shock proteins (HSPs), molecules specialized on buffering the effects of external stimuli, but which are also essential for normal cellular biochemistry. In this introductory chapter, I give a brief overview of the diversity of structure, function, and importance of these HSPs, and highlight some of the current and future research questions in this field. Additionally, this chapter acts as a bridge to the other chapters in this book. These chapters, I think you will agree, demonstrate that with regard to HSPs malaria parasites, as in so many things, obey the adage "Same same, but different."
Asunto(s)
Malaria , Parásitos , Animales , Proteínas de Choque Térmico/genética , Malaria/tratamiento farmacológico , Plasmodium falciparum/genética , Proteínas Protozoarias/genéticaRESUMEN
The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.
Asunto(s)
Azotobacter vinelandii/enzimología , Molibdeno/farmacología , Nitrogenasa/biosíntesis , Nitrogenasa/aislamiento & purificación , Azotobacter vinelandii/efectos de los fármacos , Azotobacter vinelandii/crecimiento & desarrollo , Azotobacter vinelandii/metabolismo , Biocatálisis , Medios de Cultivo/química , Relación Dosis-Respuesta a Droga , Nitrogenasa/metabolismoRESUMEN
The glucocorticoid receptor (GR) N-terminal domain (NTD) contains a transactivation domain (activation function 1; AF-1). GR AF-1 is phosphorylated, but effects of this modification upon AF-1 activity and cofactor recruitment are not completely clear. GR AF-1 activity is mostly confined to a short unstructured domain called tau1c (amino acids 187-244) that contains three phosphorylation sites and binds a short cysteine rich fragment (CH3) of the coactivator CREB binding protein (CBP). Since the CH3 domain overlaps the CBP transcriptional adaptor zinc binding (TAZ) 2 domain, implicated in phosphorylation dependent binding to other unstructured transcription factor domains, we set out to investigate whether GR interacts with TAZ2 and whether this binding event is modulated by phosphorylation. We find that GR tau1c is absolutely required for enhancement of GR function and GR/CBP association in cultured cells. Tau1c interacts with TAZ2 in vitro and peptide mapping reveals CBP binding determinants throughout tau1c. Phosphorylation at GR Ser203, not involved in transactivation, does not affect tau1c/TAZ2 interactions. However, phosphorylation at Ser211 and Ser226, markers of GR transcriptional activity, greatly enhances TAZ2 binding in a synergistic fashion. We propose that GR tau1c phosphorylation could promote CBP recruitment and enhance AF-1 activity.
Asunto(s)
Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Línea Celular , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Numerous interacting protein partners exist without recognized interactive domains, necessitating a standardized methodology to decipher more in-depth interaction profiles. Here, we present a protocol to reveal the binding partner of a secreted housekeeping enzyme, alcohol acetaldehyde dehydrogenase (Listeria adhesion protein), in Listeria monocytogenes through in silico modeling and in vivo experiments. We describe steps for target protein modeling, biophysical profiling, ClusPro docking optimization, protein variant modeling, and docking comparison. We then provide detailed procedures for in vitro and in vivo protein binding validation. For complete details on the use and execution of this protocol, please refer to Liu et al.1.
Asunto(s)
Listeria monocytogenes , Listeria , Listeria/metabolismo , Simulación por Computador , Unión ProteicaRESUMEN
Circular dichroism (CD) spectrometry is a rapid technique for detecting protein secondary structure, particularly helicity. DMSO is used to ensure optimal solubility of peptides/peptidomimetics; however, its background absorbance hinders effective CD analysis. Here, we present a protocol for reconstituting peptides/peptidomimetics from DMSO to aqueous buffers for CD analyses. We describe steps for identifying chemicals that induce DMSO evaporation, extracting peptides/peptidomimetics from DMSO, and CD spectrometer setup and analysis. We then detail procedures for secondary structure analyses of reconstituted peptides/peptidomimetics. For complete details on the use and execution of this protocol, please refer to Gao et al. (2023).1.
Asunto(s)
Dimetilsulfóxido , Peptidomiméticos , Dicroismo Circular , Dimetilsulfóxido/química , Péptidos/química , Proteínas , AguaRESUMEN
C1orf112/FIRRM is a recently identified DNA damage repair factor that regulates RAD51 in homologous recombination through interacting with the anti-recombinase FIGNL1. Here, we describe steps for purifying C1orf112/FIRRM, FIGNL1, miBRCA2, and RAD51 proteins from Escherichia coli or Saccharomyces cerevisiae cells. We then detail procedures for reconstituting the disassembly of RAD51 filament by C1orf112/FIRRM-FIGNL1 in vitro and the antagonistic effect between C1orf112/FIRRM-FIGNL1 and miBRCA2 on RAD51 filament stabilization. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2023).1.
Asunto(s)
Proteínas , Recombinasa Rad51 , Proteínas/genética , Recombinasa Rad51/genética , Reparación del ADN , Recombinación HomólogaRESUMEN
Recent advances in protein structure prediction enable 3D homology alignment and domain annotation using tertiary structures. Here, we present a protocol to identify homologous structures and annotate protein domains through in silico comparisons using the AlphaFold database. We describe steps for downloading and installing PyMOL software, preparing the query structure, and conducting a 3D homology search. The example provided highlights the application of this protocol in reevaluating an mpox viral protein annotation. For complete details on the use and execution of this protocol, please refer to Pan et al. (2023).1.
Asunto(s)
Proteínas , Programas Informáticos , Proteínas/química , Anotación de Secuencia MolecularRESUMEN
Many proteins self-assemble into dimers and higher-order oligomers. Therefore, the goal of this protocol is to characterize the conformational states of an endogenous protein of interest. Here, we present a protocol for assessing protein self-assembly in cell lysates using chemical cross-linking. We describe steps for chemical cross-linking with recombinant proteins as well as steps for cell culture and cell lysate preparation, chemical cross-linking, SDS-PAGE, and western blotting for the detection of endogenous proteins. For complete details on the use and execution of this protocol, please refer to Balaji et al.1.
Asunto(s)
Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Reactivos de Enlaces Cruzados/química , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas/química , Proteínas/metabolismo , Western Blotting/métodos , Multimerización de ProteínaRESUMEN
Palmitoylation is a post-translational lipid modification in which palmitic acid is conjugated predominantly to cysteine residues of target proteins, allowing them to tether to cell membranes. Here, we describe a protocol to perform a stepwise acyl biotin exchange assay to identify protein S-palmitoylation. We describe steps for initial blocking of free thiols in protein lysates, subsequent replacement of thioester-linked palmitate groups with a biotin tag for affinity enrichment, and identification of palmitoylated proteins by SDS-PAGE. For complete details on the use and execution of this protocol, please refer to Leishman et al.1.
Asunto(s)
Biotina , Lipoilación , Biotina/química , Biotina/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Células Cultivadas , Electroforesis en Gel de Poliacrilamida/métodosRESUMEN
Many techniques exist for the identification of protein interaction networks. We present a protocol that relies on an affinity purification-mass spectrometry (AP-MS) approach to detect proteins that co-purify with a tagged bait of interest from Drosophila melanogaster larval muscles using the GAL4/upstream activating sequence (UAS) expression system. We also describe steps for the isolation and identification of protein complexes, followed by streamlined bioinformatics analysis for rapid and reproducible results. This protocol can be extended to investigate protein interactions in other tissues. For complete details on the use and execution of this protocol, please refer to Guo et al.1.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Larva , Espectrometría de Masas , Animales , Drosophila melanogaster/metabolismo , Larva/metabolismo , Espectrometría de Masas/métodos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cromatografía de Afinidad/métodos , Mapeo de Interacción de Proteínas/métodos , Biología Computacional/métodosRESUMEN
Lysine malonylation is a protein posttranslational modification. We present a protocol to generate stable gene-knockdown K562 cell lines through lentiviral infection of a CRISPR interference (CRISPRi) system followed by lysine malonylation measurement using mass spectrometry (MS). We detail guide RNA (gRNA) vector cloning, lentiviral infection, cell line purification, protein digestion, malonyl-lysine enrichment, desalting, and MS acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Zhang et al.1 and Bons et al.2.
Asunto(s)
Lisina Acetiltransferasas , Lisina , Espectrometría de Masas , Humanos , Células K562 , Lisina/metabolismo , Espectrometría de Masas/métodos , Lisina Acetiltransferasas/metabolismo , Lisina Acetiltransferasas/genética , Sistemas CRISPR-Cas , Procesamiento Proteico-Postraduccional , Malonatos/metabolismo , ARN Guía de Sistemas CRISPR-Cas/metabolismoRESUMEN
The aggregation and spreading of "tau-seeds" are key for the development and progression of tauopathies, including Alzheimer's disease. Here we describe the steps to isolate and analyze biochemically active tau-seeds from human, mouse, and cell origin. We detail the procedure to isolate soluble tau-seeds by size exclusion chromatography and seeding assay. The isolated tau-seed can be further analyzed to determine the interactome by mass spectrometry. This workflow identifies protein-protein interactors of tau-seeds, providing a useful tool for finding new therapeutic targets. For complete details on the use and execution of this protocol, please refer to Martinez et al.1.
RESUMEN
Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.1.