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1.
Res Sq ; 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38559050

RESUMO

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.

2.
J Allergy Clin Immunol ; 153(3): 539-548, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37995859

RESUMO

The use of human antibodies as biologic therapeutics has revolutionized patient care throughout fields of medicine. As our understanding of the many roles antibodies play within our natural immune responses continues to advance, so will the number of therapeutic indications for which an mAb will be developed. The great breadth of function, long half-life, and modular structure allow for nearly limitless therapeutic possibilities. Human antibodies can be rationally engineered to enhance their desired immune functions and eliminate those that may result in unwanted effects. Antibody therapeutics now often start with fully human variable regions, either acquired from genetically engineered humanized mice or from the actual human B cells. These variable genes can be further engineered by widely used methods for optimization of their specificity through affinity maturation, random mutagenesis, targeted mutagenesis, and use of in silico approaches. Antibody isotype selection and deliberate mutations are also used to improve efficacy and tolerability by purposeful fine-tuning of their immune effector functions. Finally, improvements directed at binding to the neonatal Fc receptor can endow therapeutic antibodies with unbelievable extensions in their circulating half-life. The future of engineered antibody therapeutics is bright, with the global mAb market projected to exhibit compound annual growth, forecasted to reach a revenue of nearly half a trillion dollars in 2030.


Assuntos
Anticorpos Monoclonais , Engenharia de Proteínas , Camundongos , Animais , Humanos , Anticorpos Monoclonais/química , Engenharia de Proteínas/métodos
3.
PLoS Pathog ; 19(10): e1011496, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37871122

RESUMO

Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Anticorpos de Domínio Único , Animais , Camundongos , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Enterotoxinas/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Epitopos/metabolismo , Proteínas de Bactérias/metabolismo
4.
J Biol Chem ; 298(8): 102248, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35820485

RESUMO

Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer's disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.


Assuntos
Proteína Fosfatase 2 , Proteínas Proto-Oncogênicas c-fyn , Quinases da Família src , Doença de Alzheimer/metabolismo , Humanos , Fosfoproteínas Fosfatases , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Tirosina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo , Proteínas tau/metabolismo
5.
Pharmaceuticals (Basel) ; 15(6)2022 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-35745661

RESUMO

Background: Ergothioneine (ERGO) is a unique antioxidant and a rare amino acid available in fungi and various bacteria but not in higher plants or animals. Substantial research data indicate that ERGO is a physiological antioxidant cytoprotectant. Different from other antioxidants that need to breach the blood-brain barrier to enter the brain parenchyma, a specialized transporter called OCTN1 has been identified for transporting ERGO to the brain. Purpose: To assess whether consumption of ERGO can prevent the progress of Alzheimer's disease (AD) on young (4-month-old) 5XFAD mice. Methods and materials: Three cohorts of mice were tested in this study, including ERGO-treated 5XFAD, non-treated 5XFAD, and WT mice. After the therapy, the animals went through various behavioral experiments to assess cognition. Then, mice were scanned with PET imaging to evaluate the biomarkers associated with AD using [11C]PIB, [11C]ERGO, and [18F]FDG radioligands. At the end of imaging, the animals went through cardiac perfusion, and the brains were isolated for immunohistology. Results: Young (4-month-old) 5XFAD mice did not show a cognitive deficit, and thus, we observed modest improvement in the treated counterparts. In contrast, the response to therapy was clearly detected at the molecular level. Treating 5XFAD mice with ERGO resulted in reduced amyloid plaques, oxidative stress, and rescued glucose metabolism. Conclusions: Consumption of high amounts of ERGO benefits the brain. ERGO has the potential to prevent AD. This work also demonstrates the power of imaging technology to assess response during therapy.

6.
J Allergy Clin Immunol ; 150(6): 1525-1533, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35760390

RESUMO

BACKGROUND: Much of our understanding of the targets of IgE comes from studies of allergy, though little is known about the natural immunogenic targets seen after parasitic worm infections. OBJECTIVE: We used human monoclonal antibodies (mAbs) for an unbiased and comprehensive characterization of the immunodominant antigens targeted by IgE in conditions like allergy or helminth infection that are associated with elevated levels of IgE. METHODS: Using human hybridoma technology to immortalize IgE encoding B-cells from peripheral blood of subjects with filarial infections and elevated IgE, we generated naturally occurring human IgE mAbs. B-cell cultures were screened in an unbiased manner for IgE production without regard to specificity. Isolated IgE mAbs were then tested for binding to Brugia malayi somatic extracts using ImmunoCAP, immunoblot, and ELISA. Immunoprecipitation followed by mass spectrometry proteomics was used to identify helminth antigens that were then expressed in Escherichia coli for IgE binding characterization. RESULTS: We isolated 56 discrete IgE mAbs from 7 individuals with filarial infections. From these mAbs, we were able to definitively identify 19 filarial antigens. All IgE mAbs targeted filarial excreted/secretory proteins, including a family of previously uncharacterized proteins. Interestingly, the transthyretin-related antigens acted as the dominant inducer of the filaria-specific IgE antibody response. These filaria-specific IgE mAbs were potent inducers of anaphylaxis when passively administered to human FcεRI-expressing mice. CONCLUSIONS: We generated human hybridomas secreting naturally occurring helminth-specific IgE mAbs from filarial-infected subjects. This work provides much-needed insight into the ontogeny of helminth-induced immune response and IgE antibody response.


Assuntos
Helmintos , Hipersensibilidade , Humanos , Animais , Camundongos , Anticorpos Monoclonais
7.
PLoS Pathog ; 16(10): e1008923, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33048983

RESUMO

Type III protein secretion systems (T3SS) deliver effector proteins from the Gram-negative bacterial cytoplasm into a eukaryotic host cell through a syringe-like, multi-protein nanomachine. Cytosolic components of T3SS include a portion of the export apparatus, which traverses the inner membrane and features the opening of the secretion channel, and the sorting complex for substrate recognition and for providing the energetics required for protein secretion. Two components critical for efficient effector export are the export gate protein and the ATPase, which are proposed to be linked by the central stalk protein of the ATPase. We present the structure of the soluble export gate homo-nonamer, CdsV, in complex with the central stalk protein, CdsO, of its cognate ATPase, both derived from Chlamydia pneumoniae. This structure defines the interface between these essential T3S proteins and reveals that CdsO engages the periphery of the export gate that may allow the ATPase to catalyze an opening between export gate subunits to allow cargo to enter the export apparatus. We also demonstrate through structure-based mutagenesis of the homologous export gate in Pseudomonas aeruginosa that mutation of this interface disrupts effector secretion. These results provide novel insights into the molecular mechanisms governing active substrate recognition and translocation through a T3SS.


Assuntos
Adenosina Trifosfatases/metabolismo , Flagelos/metabolismo , Transporte Proteico/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Sistemas de Secreção Tipo III/química
8.
ACS Chem Biol ; 15(5): 1212-1221, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31909964

RESUMO

Clostridioides difficile is the leading cause of nosocomial diarrhea in the United States. The primary virulence factors are two homologous glucosyltransferase toxins, TcdA and TcdB, that inactivate host Rho-family GTPases. The glucosyltransferase activity has been linked to a "cytopathic" disruption of the actin cytoskeleton and contributes to the disruption of tight junctions and the production of pro-inflammatory cytokines. TcdB is also a potent cytotoxin that causes epithelium necrotic damage through an NADPH oxidase (NOX)-dependent mechanism. We conducted a small molecule screen to identify compounds that confer protection against TcdB-induced necrosis. We identified an enrichment of "hit compounds" with a dihydropyridine (DHP) core which led to the discovery of a key early stage calcium signal that serves as a mechanistic link between TcdB-induced NOX activation and reactive oxygen species (ROS) production. Disruption of TcdB-induced calcium signaling (with both DHP and non-DHP molecules) is sufficient to ablate ROS production and prevent subsequent necrosis in cells and in a mouse model of intoxication.


Assuntos
Anti-Infecciosos/química , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Clostridioides difficile/efeitos dos fármacos , Di-Hidropiridinas/química , Necrose/prevenção & controle , Citoesqueleto de Actina/metabolismo , Animais , Anti-Infecciosos/farmacologia , Toxinas Bacterianas/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Citocinas/metabolismo , Di-Hidropiridinas/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Glucosiltransferases/metabolismo , Humanos , Cinética , Camundongos , NADPH Oxidases/metabolismo , Necrose/induzido quimicamente , Espécies Reativas de Oxigênio/metabolismo , Fatores de Virulência/metabolismo
10.
J Biol Chem ; 293(3): 941-952, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29180448

RESUMO

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.


Assuntos
Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Enterotoxinas/metabolismo , Anticorpos Monoclonais/metabolismo , Toxinas Bacterianas/química , Células CACO-2 , Clostridioides difficile/enzimologia , Cristalografia por Raios X , Citosol/metabolismo , Enterotoxinas/química , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Rubídio/química , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismo
11.
J Biol Chem ; 292(35): 14401-14412, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28705932

RESUMO

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.


Assuntos
Antibacterianos/metabolismo , Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/enzimologia , Enterócitos/metabolismo , Enterotoxinas/metabolismo , Glucosiltransferases/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Antibacterianos/química , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Neutralizantes/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Sítios de Ligação de Anticorpos , Células CACO-2 , Sequência Conservada , Cristalografia por Raios X , Enterócitos/efeitos dos fármacos , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/toxicidade , Mapeamento de Epitopos , Glucosiltransferases/química , Glucosiltransferases/genética , Glucosiltransferases/toxicidade , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Sequências Repetitivas de Aminoácidos
12.
Artigo em Inglês | MEDLINE | ID: mdl-27512603

RESUMO

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

13.
Nat Microbiol ; 1: 15002, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-27571750

RESUMO

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.


Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Toxinas Bacterianas/metabolismo , Coenzimas/metabolismo , Cristalografia por Raios X , Enterotoxinas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Zinco/metabolismo
14.
Infect Immun ; 84(9): 2662-70, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27382020

RESUMO

Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly ß-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the ß-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the ß-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Helicobacter pylori/genética , Mutação/genética , Domínios Proteicos/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Linhagem Celular Tumoral , Células HeLa , Helicobacter pylori/metabolismo , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Microscopia Eletrônica/métodos
15.
Biochem Biophys Res Commun ; 475(1): 64-9, 2016 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-27169767

RESUMO

Alpha4 is a non-canonical regulatory subunit of Type 2A protein phosphatases that interacts directly with the phosphatase catalytic subunits (PP2Ac, PP4c, and PP6c) and is upregulated in a variety of cancers. Alpha4 modulates phosphatase expression levels and activity, but the molecular mechanism of this regulation is unclear, and the extent to which the various Type 2A catalytic subunits associate with Alpha4 is also unknown. To determine the relative fractions of the Type 2A catalytic subunits associated with Alpha4, we conducted Alpha4 immunodepletion experiments in HEK293T cells and found that a significant fraction of total PP6c is associated with Alpha4, whereas a minimal fraction of total PP2Ac is associated with Alpha4. To facilitate studies of phosphatases in the presence of mutant or null Alpha4 alleles, we developed a facile and rapid method to simultaneously knockdown and rescue Alpha4 in tissue culture cells. This approach has the advantage that levels of endogenous Alpha4 are dramatically reduced by shRNA expression thereby simplifying interpretation of mutant phenotypes. We used this system to show that knockdown of Alpha4 preferentially impacts the expression of PP4c and PP6c compared to expression levels of PP2Ac.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Domínio Catalítico , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/genética , Chaperonas Moleculares , Fosfoproteínas Fosfatases/análise , Proteína Fosfatase 2/análise
16.
Proc Natl Acad Sci U S A ; 112(30): 9346-51, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26170302

RESUMO

Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. Here, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.


Assuntos
Anticorpos Antivirais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Sítios de Ligação , Cristalografia por Raios X , Variação Genética , Humanos , Fragmentos de Imunoglobulinas/química , Influenza Humana/imunologia , Conformação Molecular , Dados de Sequência Molecular , Mutação , Ácido N-Acetilneuramínico/química , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
17.
PLoS Pathog ; 10(11): e1004498, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25375170

RESUMO

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.


Assuntos
Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos , Chlamydia/química , Chaperonas Moleculares/química , Estrutura Quaternária de Proteína , Transporte Proteico , Shigella/química
18.
J Biol Chem ; 289(42): 29273-84, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25164821

RESUMO

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.


Assuntos
Glutamil Aminopeptidase/metabolismo , Rim/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Pressão Sanguínea , Clonagem Molecular , Humanos , Medula Renal/metabolismo , Espectrometria de Massas , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Oócitos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Sódio/metabolismo , Xenopus laevis
19.
J Virol ; 88(1): 469-76, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24155406

RESUMO

Previous human antibody studies have shown that the human VH1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação de Anticorpos , Biopolímeros/imunologia , Microscopia Crioeletrônica , Primers do DNA , Epitopos/química , Espectrometria de Massas , Dados de Sequência Molecular
20.
J Clin Invest ; 123(10): 4405-9, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23999429

RESUMO

Recent studies described the experimental adaptation of influenza H5 HAs that confers respiratory droplet transmission (rdt) to influenza virus in ferrets. Acquisition of the ability to transmit via aerosol may lead to the development of a highly pathogenic pandemic H5 virus. Vaccines are predicted to play an important role in H5N1 control should the virus become readily transmissible between humans. We obtained PBMCs from patients who received an A/Vietnam/1203/2004 H5N1 subunit vaccine. Human hybridomas were then generated and characterized. We identified antibodies that bound the HA head domain and recognized both WT and rdt H5 HAs. We used a combination of structural techniques to define a mechanism of antibody recognition of an H5 HA receptor-binding site that neutralized H5N1 influenza viruses and pseudoviruses carrying the HA rdt variants that have mutations near the receptor-binding site. Incorporation or retention of this critical antigenic site should be considered in the design of novel H5 HA immunogens to protect against mammalian-adapted H5N1 mutants.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/prevenção & controle , Vacinação , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Mapeamento de Epitopos , Humanos , Hibridomas , Influenza Humana/transmissão , Influenza Humana/virologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
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