RESUMO
BTN2 gene expression in the yeast Saccharomyces cerevisiae is up-regulated in response to the deletion of BTN1, which encodes the ortholog of a human Batten disease protein. We isolated Btn2 as a Snc1 v-SNARE binding protein using the two-hybrid assay and examined its role in intracellular protein trafficking. We show that Btn2 is an ortholog of the Drosophila and mammalian Hook1 proteins that interact with SNAREs, cargo proteins, and coat components involved in endosome-Golgi protein sorting. By immunoprecipitation, it was found that Btn2 bound the yeast endocytic SNARE complex (e.g., Snc1 and Snc2 [Snc1/2], Tlg1, Tlg2, and Vti1), the Snx4 sorting nexin, and retromer (e.g., Vps26 and Vps35). In in vitro binding assays, recombinant His(6)-tagged Btn2 bound glutathione S-transferase (GST)-Snc1 and GST-Vps26. Btn2-green fluorescent protein and Btn2-red fluorescent protein colocalize with Tlg2, Snx4, and Vps27 to a compartment adjacent to the vacuole that corresponds to a late endosome. The deletion of BTN2 blocks Yif1 retrieval back to the Golgi apparatus, while the localization of Ste2, Fur4, Snc1, Vps10, carboxypeptidases Y (CPY) and S (CPS), Sed5, and Sec7 is unaltered in btn2Delta cells. Yif1 delivery to the vacuole was observed in other late endosome-Golgi trafficking mutants, including ypt6Delta, snx4Delta, and vps26Delta cells. Thus, Btn2 facilitates specific protein retrieval from a late endosome to the Golgi apparatus, a process which may be adversely affected in patients with Batten disease.
Assuntos
Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Mutação , Lipofuscinoses Ceroides Neuronais/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas R-SNARE/metabolismo , Proteínas SNARE/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismoRESUMO
Gcs1 is an Arf GTPase-activating protein (Arf-GAP) that mediates Golgi-ER and post-Golgi vesicle transport in yeast. Here we show that the Snc1,2 v-SNAREs, which mediate endocytosis and exocytosis, interact physically and genetically with Gcs1. Moreover, Gcs1 and the Snc v-SNAREs colocalize to subcellular structures that correspond to the trans-Golgi and endosomal compartments. Studies performed in vitro demonstrate that the Snc-Gcs1 interaction results in the efficient binding of recombinant Arf1Delta17N-Q71L to the v-SNARE and the recruitment of purified coatomer. In contrast, the presence of Snc had no effect on Gcs1 Arf-GAP activity in vitro, suggesting that v-SNARE binding does not attenuate Arf1 function. Disruption of both the SNC and GCS1 genes results in synthetic lethality, whereas overexpression of either SNC gene inhibits the growth of a distinct subset of COPI mutants. We show that GFP-Snc1 recycling to the trans-Golgi is impaired in gcs1Delta cells and these COPI mutants. Together, these results suggest that Gcs1 facilitates the incorporation of the Snc v-SNAREs into COPI recycling vesicles and subsequent endosome-Golgi sorting in yeast.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Proteínas R-SNARE/metabolismo , Leveduras/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Endocitose , Endossomos , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Proteínas Ativadoras de GTPase/genética , Genes Fúngicos , Genes Letais , Imunoprecipitação , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteínas R-SNARE/análise , Proteínas R-SNARE/genética , Deleção de Sequência , Técnicas do Sistema de Duplo-Híbrido , Leveduras/químicaRESUMO
OBJECTIVE: Timely publication of data is important for the medical community and provides a valuable contribution to data disclosure. The objective of this study was to identify and evaluate times to acceptance and publication for peer-reviewed manuscripts, reviews, and letters to the editor. RESEARCH DESIGN AND METHODS: Key publication metrics for published manuscripts, reviews, and letters to the editor were identified by eight Amgen publications professionals. Data for publications submitted between 1 January 2013 and 1 November 2015 were extracted from a proprietary internal publication-tracking database. Variables included department initiating the study, publication type, number of submissions per publication, and the total number of weeks from first submission to acceptance, online publication, and final publication. RESULTS: A total of 337 publications were identified, of which 300 (89%) were manuscripts. Time from submission to acceptance and publication was generally similar between clinical and real-world evidence (e.g. observational and health economics studies) publications. Median (range) time from first submission to acceptance was 23.4 (0.2-226.2) weeks. Median (range) time from first submission to online (early-release) publication was 29.7 (2.4-162.6) weeks. Median (range) time from first submission to final (print) publication was 36.2 (2.8-230.8) weeks. Time from first submission to acceptance, online publication, and final publication increased accordingly with number of submissions required for acceptance, with similar times noted between each subsequent submission. CONCLUSIONS: Analysis of a single-company publication database showed that the median time for manuscripts to be fully published after initial submission was 36.2 weeks, and time to publication increased accordingly with the number of submissions. Causes for multiple submissions and time from clinical trial completion to first submission were not assessed; these were limitations of the study. Nonetheless, publication planners should consider these results when evaluating timelines and identifying potential journals early in the publication planning process.
Assuntos
Revisão da Pesquisa por Pares , Publicações/estatística & dados numéricos , Editoração/estatística & dados numéricos , Humanos , Fatores de TempoRESUMO
OBJECTIVE: The Physician Payments Sunshine Act, enacted in 2010, is intended to increase the transparency of relationships between US physicians and teaching hospitals and manufacturers of drugs, biologics, and medical devices. We examined current opinion regarding the impact of the Sunshine Act on peer-reviewed medical publications. RESEARCH DESIGN AND METHODS: We searched indexed databases (NLM/PubMed, EMBASE, and Scopus) and nonindexed sources (lay and medical press, medical websites, congress abstracts) for articles published between January 2010 and June 2015 that contained terms indicative of content related to the Sunshine Act (e.g., 'Sunshine Act', 'open payment program'). Nine publication professionals then systematically reviewed identified articles for publications-related content. MAIN OUTCOME MEASURES: Quantification and characterization of publications that focused on the Sunshine Act and its implications for medical publishing. RESULTS: Among 1200 indexed publications, 113 had content on the Sunshine Act. Thirty-one discussed its implications for publications; nine distinguished between financial and nonfinancial transfers of value. Of the 117 nonindexed publications with content on the Sunshine Act, 16 discussed implications for publications, and seven distinguished between financial and nonfinancial transfers of value. Reporting of such transfers of value was viewed as a potential barrier to participation in publications with industry support. CONCLUSIONS: There is limited literature on the impact of the Sunshine Act on peer-reviewed publications and limited physician awareness that publication support may be reported as a transfer of value.
Assuntos
Revisão da Pesquisa por Pares/normas , Médicos/normas , Hospitais de Ensino/normas , HumanosRESUMO
IMPORTANCE OF THE FIELD: Improving the pharmacodynamics of protein drugs has the potential to improve the care and the quality of life of patients suffering from a variety of diseases. AREAS COVERED IN THIS REVIEW: Four approaches to improve protein drugs are described: PEGylation, amino acid substitution, fusion to carrier proteins and encapsulation. A new platform technology based on the binding of proteins/peptides to the outer surface of PEGylated liposomes (PEGLip) is then presented. Binding of proteins to PEGLip is non-covalent, highly specific and dependent on an amino acid consensus sequence within the proteins. Association of proteins with PEGLip results in substantial enhancement of the pharmacodynamic properties of proteins following administration. This has been demonstrated in preclinical studies and clinical trials with coagulation factors VIII and VIIa. It has also been demonstrated in preclinical studies with granulocyte colony-stimulating factor. A mechanism is presented that explains the improvements in hemostatic efficacy of PEGLip-formulated coagulation factors VIII and VIIa. WHAT THE READER WILL GAIN: The reader will gain an understanding of the advantages and disadvantages of each of the approaches discussed. TAKE HOME MESSAGE: PEGLip formulation is an important new approach to improve the pharmacodynamics of protein drugs. This approach may be applied to further therapeutic proteins in the future.
Assuntos
Química Farmacêutica/métodos , Fator VIII/administração & dosagem , Fator VIIa/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Lipossomos/síntese química , Proteínas/administração & dosagem , Animais , Anticoagulantes/administração & dosagem , Avaliação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos , Lipossomos/administração & dosagem , Modelos Biológicos , Modelos Moleculares , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Proteínas/químicaRESUMO
Hemophilia A is a rare X-linked bleeding disorder caused by lack or dysfunction of coagulation factor VIII (FVIII). Hemophilia A is treated with replacement therapy, but frequent injections of the missing FVIII often lead to the formation of inhibitory antibodies. Patients who develop high levels of inhibitors must be treated with bypassing agents such as activated FVII (FVIIa). Both FVIII and FVIIa have short half-lives and require multiple injections. Long-acting forms of these proteins would therefore reduce the frequency of injections, improve patient compliance and reduce complications. In this article we present a new platform technology that produces long-acting forms of FVIII and FVIIa and improves the efficacy of hemophilia treatment. This technology is based on the binding of proteins/peptides to the outer surface of PEGylated liposomes (PEGLip). Binding is dependent on an amino acid consensus sequence within the proteins and is highly specific. At the same time, binding is non-covalent and does not require any modification of the therapeutic agent or its production process. Association of proteins with PEGLip results in substantial enhancements in their pharmacodynamic properties following administration. These improvements seem to arise from the association of formulated proteins with platelets prior to induction of coagulation.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fator VIII/administração & dosagem , Fator VIIa/administração & dosagem , Hemofilia A/tratamento farmacológico , Sequência de Aminoácidos , Animais , Química Farmacêutica , Sequência Consenso , Preparações de Ação Retardada , Modelos Animais de Doenças , Fator VIII/genética , Fator VIIa/genética , Hemofilia A/sangue , Humanos , Lipossomos/química , Camundongos , Nanomedicina , Polietilenoglicóis/químicaRESUMO
We have previously shown that formulation of coagulation factor VIII and activated factor VII with PEGylated liposomes (PEGLip) results in an extension of circulation time and an increase in hemostatic efficacy. Here we identified additional proteins that associate with PEGLip, including granulocyte colony-stimulating factor (G-CSF). Surface plasmon resonance analyses indicated that G-CSF bound noncovalently but with high affinity and specificity to PEGLip. A pharmacokinetic study in mice demonstrated that PEGLip formulation of G-CSF extended its circulation time and resulted in higher G-CSF levels several hours after both subcutaneous and intravenous injection. PEGLip-formulated G-CSF had a significantly improved efficacy in the mobilization of hematopoietic stem cells (HSC) from the bone marrow to the peripheral blood. The results suggest that PEGLip-formulated G-CSF may function as an effective and safe tool for the mobilization of HSC prior to bone marrow transplantation. We also identified an amino acid sequence present in proteins that associate with PEGLip but absent from those that do not. A peptide based on this consensus sequence bound PEGLip. The results suggest that PEGLip formulation may serve as a platform for the delivery of additional short-half-life proteins/peptides having the relevant consensus sequence.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Polietilenoglicóis/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Consenso , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/química , Humanos , Injeções Intravenosas , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ligação Proteica , Proteínas RecombinantesRESUMO
The yeast exocytic SNARE complex consists of one molecule each of the Sso1/2 target SNAREs, Snc1/2 vesicular SNAREs, and the Sec9 target SNARE, which form a fusion complex that is conserved in evolution. Another protein, Sec1, binds to the SNARE complex to facilitate assembly. We show that Mso1, a Sec1-interacting protein, also binds to the SNARE complex and plays a role in mediating Sec1 functions. Like Sec1, Mso1 bound to SNAREs in cells containing SNARE complexes (i.e. wild-type, sec1-1, and sec18-1 cells), but not in cells in which complex formation is inhibited (i.e. sec4-8 cells). Nevertheless, Mso1 remained associated with Sec1 even in sec4-8 cells, indicating that they act as a pair. Mso1 localized primarily to the plasma membrane of the bud when SNARE complex formation was not impaired but was mostly in the cytoplasm when assembly was prevented. Genetic studies suggest that Mso1 enhances Sec1 function while attenuating Sec4 GTPase function. This dual action may impart temporal regulation between Sec4 turnoff and Sec1-mediated SNARE assembly. Notably, a small region at the C terminus of Mso1 is conserved in the mammalian Munc13/Mint proteins and is necessary for proper membrane localization. Overexpression of Mso1 lacking this domain (Mso1-(1-193)) inhibited the growth of cells bearing an attenuated Sec4 GTPase. These results suggest that Mso1 is a component of the exocytic SNARE complex and a possible ortholog of the Munc13/Mint proteins.