RESUMO
Orientia tsutsugamushi is an etiologic agent of scrub typhus, a globally emerging rickettsiosis that can be fatal. The bacterium's obligate intracellular lifestyle requires its interaction with host eukaryotic cellular pathways. The proteins it employs to do so and their functions during infection are understudied. Recombinant versions of the recently characterized O. tsutsugamushi deubiquitylase (OtDUB) exhibit high-affinity ubiquitin binding, mediate guanine nucleotide exchange to activate Rho GTPases, bind clathrin adaptor protein complexes 1 and 2, and bind the phospholipid phosphatidylserine. Whether OtDUB is expressed and its function during O. tsutsugamushi infection have yet to be explored. Here, OtDUB expression, location, and interactome during infection were examined. O. tsutsugamushi transcriptionally and translationally expresses OtDUB throughout infection of epithelial, monocytic, and endothelial cells. Results from structured illumination microscopy, surface trypsinization of intact bacteria, and acetic acid extraction of non-integral membrane proteins indicate that OtDUB peripherally associates with the O. tsutsugamushi cell wall and is at least partially present on the bacterial surface. Analyses of the proteins with which OtDUB associates during infection revealed several known O. tsutsugamushi cell wall proteins and others. It also forms an interactome with adapter protein complex 2 and other endosomal membrane traffic regulators. This study documents the first interactors of OtDUB during O. tsutsugamushi infection and establishes a strong link between OtDUB and the host endocytic pathway.
Assuntos
Orientia tsutsugamushi , Tifo por Ácaros , Humanos , Orientia tsutsugamushi/fisiologia , Células Endoteliais , Ligação Proteica , MonócitosRESUMO
Host cell membrane-trafficking pathways are often manipulated by bacterial pathogens to gain cell entry, avoid immune responses, or to obtain nutrients. The 1,369-residue OtDUB protein from the obligate intracellular human pathogen Orientia tsutsugamushi bears a deubiquitylase (DUB) and additional domains. Here we show that OtDUB ectopic expression disrupts membrane trafficking through multiple mechanisms. OtDUB binds directly to the clathrin adaptor-protein (AP) complexes AP-1 and AP-2, and the OtDUB275-675 fragment is sufficient for binding to either complex. To assess the impact of OtDUB interactions with AP-1 and AP-2, we examined trans-Golgi trafficking and endocytosis, respectively. Endocytosis is reduced by two separate OtDUB fragments: one contains the AP-binding domain (OtDUB1-675), and the other does not (OtDUB675-1369). OtDUB1-675 disruption of endocytosis requires its ubiquitin-binding capabilities. OtDUB675-1369 also fragments trans- and cis-Golgi structures. Using a growth-based selection in yeast, we identified viable OtDUB675-1369 point mutants that also no longer caused Golgi defects in human cells. In parallel, we found OtDUB675-1369 binds directly to phosphatidylserine, and this lipid binding is lost in the same mutants. Together these results show that OtDUB contains multiple activities capable of modulating membrane trafficking. We discuss how these activities may contribute to Orientia infections.
Assuntos
Orientia tsutsugamushi , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Endocitose , Complexo de Golgi/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Orientia tsutsugamushi/metabolismo , Ligação Proteica , Tifo por Ácaros/metabolismo , Tifo por Ácaros/microbiologia , Tifo por Ácaros/patologiaRESUMO
Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.
Assuntos
Proteínas de Saccharomyces cerevisiae , Ubiquitina , Humanos , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Wolbachia bacteria, inherited through the female germ line, infect a large fraction of arthropod species. Many Wolbachia strains manipulate host reproduction, most commonly through cytoplasmic incompatibility (CI). CI, a conditional male sterility, results when Wolbachia-infected male insects mate with uninfected females; viability is restored if the female is similarly infected (called "rescue"). CI is used to help control mosquito-borne viruses such as dengue and Zika, but its mechanisms remain unknown. The coexpressed CI factors CifA and CifB form stable complexes in vitro, but the timing and function of this interaction in the insect are unresolved. CifA expression in the female germ line is sufficient for rescue. We report high-resolution structures of a CI-factor complex, CinA-CinB, which utilizes a unique binding mode between the CinA rescue factor and the CinB nuclease; the structures were validated by biochemical and yeast growth analyses. Importantly, transgenic expression in Drosophila of a nonbinding CinA mutant, designed based on the CinA-CinB structure, suggests CinA expressed in females must bind CinB imported by sperm in order to rescue embryonic viability. Binding between cognate factors is conserved in an enzymatically distinct CI system, CidA-CidB, suggesting universal features in Wolbachia CI induction and rescue.
Assuntos
Drosophila melanogaster/microbiologia , Embrião não Mamífero/embriologia , Infertilidade Masculina/fisiopatologia , Reprodução/fisiologia , Wolbachia/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/genética , Desenvolvimento Embrionário , Feminino , Masculino , Controle de Mosquitos/métodos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Simbiose , Doenças Transmitidas por Vetores/prevenção & controle , Doenças Transmitidas por Vetores/transmissão , Doenças Transmitidas por Vetores/virologiaRESUMO
Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEF alone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.
Assuntos
Proteínas de Bactérias/química , Fatores de Troca do Nucleotídeo Guanina/química , Modelos Moleculares , Orientia tsutsugamushi , Sítios de Ligação , Cristalografia por Raios X , Complexos Multiproteicos , Orientia tsutsugamushi/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Tifo por Ácaros/microbiologia , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Ubiquitin mediated signaling contributes critically to host cell defenses during pathogen infection. Many pathogens manipulate the ubiquitin system to evade these defenses. Here we characterize a likely effector protein bearing a deubiquitylase (DUB) domain from the obligate intracellular bacterium Orientia tsutsugamushi, the causative agent of scrub typhus. The Ulp1-like DUB prefers ubiquitin substrates over ubiquitin-like proteins and efficiently cleaves polyubiquitin chains of three or more ubiquitins. The co-crystal structure of the DUB (OtDUB) domain with ubiquitin revealed three bound ubiquitins: one engages the S1 site, the second binds an S2 site contributing to chain specificity and the third binds a unique ubiquitin-binding domain (UBD). The UBD modulates OtDUB activity, undergoes a pronounced structural transition upon binding ubiquitin, and binds monoubiquitin with an unprecedented ~5 nM dissociation constant. The characterization and high-resolution structure determination of this enzyme should aid in its development as a drug target to counter Orientia infections.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Orientia tsutsugamushi/enzimologia , Tifo por Ácaros/microbiologia , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Lisina/metabolismo , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , TermodinâmicaRESUMO
The E3 ligase membrane-associated ring-CH-type finger 6 (MARCH6) is a polytopic enzyme bound to the membranes of the endoplasmic reticulum. It controls levels of several known protein substrates, including a key enzyme in cholesterol synthesis, squalene monooxygenase. However, beyond its own autodegradation, little is known about how MARCH6 itself is regulated. Using CRISPR/Cas9 gene-editing, MARCH6 overexpression, and immunoblotting, we found here that cholesterol stabilizes MARCH6 protein endogenously and in HEK293 cells that stably express MARCH6. Conversely, MARCH6-deficient HEK293 and HeLa cells lost their ability to degrade squalene monooxygenase in a cholesterol-dependent manner. The ability of cholesterol to boost MARCH6 did not seem to involve a putative sterol-sensing domain in this E3 ligase, but was abolished when either membrane extraction by valosin-containing protein (VCP/p97) or proteasomal degradation was inhibited. Furthermore, cholesterol-mediated stabilization was absent in two MARCH6 mutants that are unable to degrade themselves, indicating that cholesterol stabilizes MARCH6 protein by preventing its autodegradation. Experiments with chemical chaperones suggested that this likely occurs through a conformational change in MARCH6 upon cholesterol addition. Moreover, cholesterol reduced the levels of at least three known MARCH6 substrates, indicating that cholesterol-mediated MARCH6 stabilization increases its activity. Our findings highlight an important new role for cholesterol in controlling levels of proteins, extending the known repertoire of cholesterol homeostasis players.
Assuntos
Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Sistemas CRISPR-Cas , Colesterol/genética , Ativação Enzimática/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Ubiquitina-Proteína Ligases/genética , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
A family of virulence factors from the bacterial pathogen Legionella pneumophila has been discovered to modify human Rab GTPases with ubiquitin. Surprisingly, this modification occurs via a non-canonical mechanism that uses nicotinamide adenine dinucleotide as a cofactor.
Assuntos
Legionella pneumophila/patogenicidade , Doença dos Legionários/enzimologia , Doença dos Legionários/microbiologia , Ubiquitina/metabolismo , Fatores de Virulência/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Legionella pneumophila/isolamento & purificação , Legionella pneumophila/metabolismo , NAD/metabolismo , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Sequência Conservada/genética , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Immunoblotting , Proteínas de Membrana/genética , Microscopia de Fluorescência , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
The inner membrane of the nuclear envelope (NE) is home to hundreds of integral membrane proteins (NE transmembrane proteins, "NETs") with conserved or tissue-specific roles in genome organization and nuclear function. Nearly all characterized NETs bind A- or B-type lamins directly. However, hundreds of NETs remain uncharacterized, collectively posing an enormous gap that must be bridged to understand nuclear function and genome biology. We provide technically simple protocols for the separation and recovery of functionally distinct populations of NETs and A-type lamins. This protocol was developed for emerin, an inner nuclear membrane protein that binds lamins and barrier-to-autointegration factor (BANF1) as a component of nuclear lamina structure, and has diverse roles in nuclear assembly, signaling, and gene regulation. This protocol separates easily solubilized ("easy") populations of nuclear lamina proteins (emerin, lamin A, BAF) from "sonication-dependent" populations. Depending on cell type, the "easy" and "sonication-dependent" fractions each contain up to about half the available emerin, A-type lamins, and BAF, whereas B-type lamins and histone H3 are predominantly sonication dependent. The two populations of emerin have distinct posttranslational modifications, and only one population associates with BAF. This method may be useful for functional screening or analysis of other lamin-associated proteins, including novel NETs emerging from proteomic studies.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/isolamento & purificação , Fracionamento Celular , Centrifugação , Células HEK293 , Células HeLa , Humanos , ImunoprecipitaçãoRESUMO
Emerin is a conserved membrane component of nuclear lamina structure. Here, we report an advance in understanding the molecular basis of emerin function: intermolecular emerin-emerin association. There were two modes: one mediated by association of residues 170-220 in one emerin molecule to residues 170-220 in another, and the second involving residues 170-220 and 1-132. Deletion analysis showed residues 187-220 contain a positive element essential for intermolecular association in cells. By contrast, deletion of residues 168-186 inactivated a proposed negative element, required to limit or control association. Association of GFP-emerin with nuclear BAF in cells required the LEM domain (residues 1-47) and the positive element. Emerin peptide arrays revealed direct binding of residues 170-220 to residues 206-225 (the proposed positive element), residues 147-174 (particularly P(153)MYGRDSAYQSITHYRP(169)) and the LEM domain. Emerin residues 1-132 and 159-220 were each sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins. These results, and predicted emerin intrinsic disorder, support the hypothesis that there are multiple 'backbone' and LEM-domain configurations in a proposed intermolecular emerin network at the nuclear envelope.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Lâmina Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Lâmina Nuclear/química , Lâmina Nuclear/genética , Proteínas Nucleares/genética , Ligação ProteicaRESUMO
Emerin, a membrane component of nuclear "lamina" networks with lamins and barrier to autointegration factor (BAF), is highly O-GlcNAc-modified ("O-GlcNAcylated") in mammalian cells. Mass spectrometry analysis revealed eight sites of O-GlcNAcylation, including Ser-53, Ser-54, Ser-87, Ser-171, and Ser-173. Emerin O-GlcNAcylation was reduced ~50% by S53A or S54A mutation in vitro and in vivo. O-GlcNAcylation was reduced ~66% by the triple S52A/S53A/S54A mutant, and S173A reduced O-GlcNAcylation of the S52A/S53A/S54A mutant by ~30%, in vivo. We separated two populations of emerin, A-type lamins and BAF; one population solubilized easily, and the other required sonication and included histones and B-type lamins. Emerin and BAF associated only in histone- and lamin-B-containing fractions. The S173D mutation specifically and selectively reduced GFP-emerin association with BAF by 58% and also increased GFP-emerin hyper-phosphorylation. We conclude that ß-N-acetylglucosaminyltransferase, an essential enzyme, controls two regions in emerin. The first region, defined by residues Ser-53 and Ser-54, flanks the LEM domain. O-GlcNAc modification at Ser-173, in the second region, is proposed to promote emerin association with BAF in the chromatin/lamin B "niche." These results reveal direct control of a conserved LEM domain nuclear lamina component by ß-N-acetylglucosaminyltransferase, a nutrient sensor that regulates cell stress responses, mitosis, and epigenetics.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Laminina/metabolismo , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Lâmina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Acetilglucosamina , Acilação/fisiologia , Substituição de Aminoácidos , Cromatina/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Laminina/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , N-Acetilglucosaminiltransferases/genética , Lâmina Nuclear/genética , Proteínas Nucleares/genética , Fosforilação/fisiologiaRESUMO
Emerin, a conserved LEM-domain protein, is among the few nuclear membrane proteins for which extensive basic knowledge--biochemistry, partners, functions, localizations, posttranslational regulation, roles in development and links to human disease--is available. This review summarizes emerin and its emerging roles in nuclear "lamina" structure, chromatin tethering, gene regulation, mitosis, nuclear assembly, development, signaling and mechano-transduction. We also highlight many open questions, exploration of which will be critical to understand how this intriguing nuclear membrane protein and its "family" influence the genome.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animais , Humanos , Proteínas de Membrana/genética , Proteínas Nucleares/genéticaRESUMO
Because cell-division failure is deleterious, promoting tumorigenesis in mammals, cells utilize numerous mechanisms to control their cell-cycle progression. Though cell division is considered a well-ordered sequence of biochemical events, cytokinesis, an inherently mechanical process, must also be mechanically controlled to ensure that two equivalent daughter cells are produced with high fidelity. Given that cells respond to their mechanical environment, we hypothesized that cells utilize mechanosensing and mechanical feedback to sense and correct shape asymmetries during cytokinesis. Because the mitotic spindle and myosin II are vital to cell division, we explored their roles in responding to shape perturbations during cell division. We demonstrate that the contractile proteins myosin II and cortexillin I redistribute in response to intrinsic and externally induced shape asymmetries. In early cytokinesis, mechanical load overrides spindle cues and slows cytokinesis progression while contractile proteins accumulate and correct shape asymmetries. In late cytokinesis, mechanical perturbation also directs contractile proteins but without apparently disrupting cytokinesis. Significantly, this response only occurs during anaphase through cytokinesis, does not require microtubules, and is independent of spindle orientation, but is dependent on myosin II. Our data provide evidence for a mechanosensory system that directs contractile proteins to regulate cell shape during mitosis.