RESUMO
Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo-inositol's molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol's primary target was not the native, unfolded, or final aggregated states of the protein; rather, we propose that it was the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.
Assuntos
Catarata , Cristalino , Catarata/metabolismo , Humanos , Inositol/análise , Inositol/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , Agregados ProteicosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.
Assuntos
Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/farmacologia , Imunidade Humoral/efeitos dos fármacos , Peptídeos/imunologia , Engenharia de Proteínas , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antígenos/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Endocitose/efeitos dos fármacos , Epitopos/imunologia , Imunização , Memória Imunológica/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/química , Peptídeos/química , Fosfosserina/metabolismoRESUMO
When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Sinapses Imunológicas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Movimento Celular , Citoesqueleto/metabolismo , Humanos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Linfócitos T/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
TorsinA is an ER-resident AAA + ATPase, whose deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is an unusual AAA + ATPase that needs an external activator. Also, it likely does not thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of its still enigmatic function. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 Å structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner channel of ~ 4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA + ATPases. We discuss the implications of these observations for TorsinA function.
Assuntos
Adenosina Trifosfatases/química , Modelos Moleculares , Chaperonas Moleculares/química , Polímeros/química , Conformação Proteica , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Polimerização , Polímeros/metabolismoRESUMO
Sphingolipids are structural membrane components and important eukaryotic signaling molecules. Sphingolipids regulate inflammation and immunity and were recently identified as the most differentially abundant metabolite in stool from inflammatory bowel disease (IBD) patients. Commensal bacteria from the Bacteroidetes phylum also produce sphingolipids, but the impact of these metabolites on host pathways is largely uncharacterized. To determine whether bacterial sphingolipids modulate intestinal health, we colonized germ-free mice with a sphingolipid-deficient Bacteroides thetaiotaomicron strain. A lack of Bacteroides-derived sphingolipids resulted in intestinal inflammation and altered host ceramide pools in mice. Using lipidomic analysis, we described a sphingolipid biosynthesis pathway and revealed a variety of Bacteroides-derived sphingolipids including ceramide phosphoinositol and deoxy-sphingolipids. Annotating Bacteroides sphingolipids in an IBD metabolomic dataset revealed lower abundances in IBD and negative correlations with inflammation and host sphingolipid production. These data highlight the role of bacterial sphingolipids in maintaining homeostasis and symbiosis in the gut.
Assuntos
Bacteroides thetaiotaomicron/crescimento & desenvolvimento , Bacteroides thetaiotaomicron/metabolismo , Interações entre Hospedeiro e Microrganismos , Intestinos/microbiologia , Intestinos/fisiologia , Esfingolipídeos/metabolismo , Simbiose/efeitos dos fármacos , Animais , Vida Livre de Germes , Homeostase/efeitos dos fármacos , Doenças Inflamatórias Intestinais/prevenção & controle , Intestinos/efeitos dos fármacos , CamundongosRESUMO
We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-ß inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles' cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8+ T cells relative to untargeted particles, and delivery of TGF-ß inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases.
Assuntos
Sistemas de Liberação de Medicamentos , Ouro/química , Imunoconjugados/química , Nanopartículas Metálicas/química , Bibliotecas de Moléculas Pequenas/administração & dosagem , Linfócitos T/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Células Cultivadas , Feminino , Ouro/farmacocinética , Imunoconjugados/farmacocinética , Camundongos Endogâmicos C57BL , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/análiseRESUMO
Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs.
Assuntos
Endossomos/imunologia , Proteínas de Membrana Transportadoras/imunologia , Chaperonas Moleculares/imunologia , Receptores Toll-Like/imunologia , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células HEK293 , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estabilidade Proteica , Transporte Proteico/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células THP-1 , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.
Assuntos
Colágeno Tipo I/química , Proteínas de Choque Térmico HSP90/química , Ensaios de Triagem em Larga Escala , Glicoproteínas de Membrana/química , Benzoquinonas/farmacologia , Linhagem Celular , Colágeno Tipo I/biossíntese , Colágeno Tipo I/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Isoformas de Proteínas/químicaRESUMO
The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions. Analyses of pooled and individual mutants reveal the importance of the high-affinity phosphate transport system (the Pst system), PhoR, and glycolipid and ornithine lipid synthases during phosphate limitation. The phosphate-dependent synthesis of MGDG in G20 and the widespread occurrence of the MGDG/GADG synthase among sulfate reducing ∂-Proteobacteria implicate these microbes in the production of abundant MGDG in anaerobic environments where the concentrations of phosphate are lower than 10 µM. Numerous predicted changes in the composition of the cell envelope and systems involved in transport, maintenance of cytoplasmic redox potential, central metabolism and regulatory pathways also suggest an impact of phosphate limitation on the susceptibility of sulfate reducing bacteria to other anthropogenic or environmental stresses.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Desulfovibrio/efeitos dos fármacos , Desulfovibrio/fisiologia , Fosfatos/farmacologia , Aclimatação/efeitos dos fármacos , Anaerobiose , Desulfovibrio/citologia , Desulfovibrio/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Mutação , Fósforo/metabolismoRESUMO
Erythrocytes are attractive as potential cell-based drug carriers because of their abundance and long lifespan in vivo. Existing methods for loading drug cargos into erythrocytes include hypotonic treatments, electroporation, and covalent attachment onto the membrane, all of which require ex vivo manipulation. Here, we characterized the properties of amphiphilic gold nanoparticles (amph-AuNPs), comprised of a â¼2.3 nm gold core and an amphiphilic ligand shell, which are able to embed spontaneously within erythrocyte membranes and might provide a means to load drugs into red blood cells (RBCs) directly in vivo. Particle interaction with RBC membranes occurred rapidly at physiological temperature. We further show that amph-AuNP uptake by RBCs was limited by the glycocalyx and was particularly influenced by sialic acids on cell surface proteoglycans. Using a reductionist model membrane system with synthetic lipid vesicles, we confirmed the importance of membrane fluidity and the glycocalyx in regulating amph-AuNP/membrane interactions. These results thus provide evidence for the interaction of amph-AuNPs with erythrocyte membranes and identify key membrane components that govern this interaction, providing a framework for the development of amph-AuNP-carrying erythrocyte 'pharmacytes' in vivo.
Assuntos
Membrana Eritrocítica/química , Membrana Eritrocítica/ultraestrutura , Glicocálix/química , Glicocálix/ultraestrutura , Ouro/química , Nanopartículas Metálicas/química , Humanos , Nanopartículas Metálicas/ultraestruturaRESUMO
Conversion of carbohydrates to lipids at high yield and productivity is essential for cost-effective production of renewable biodiesel. Although some microorganisms can convert sugars to oils, conversion yields and rates are typically low due primarily to allosteric inhibition of the lipid biosynthetic pathway by saturated fatty acids. By reverse engineering the mammalian cellular obese phenotypes, we identified the delta-9 stearoyl-CoA desaturase (SCD) as a rate limiting step and target for the metabolic engineering of the lipid synthesis pathway in Yarrowia lipolytica. Simultaneous overexpression of SCD, Acetyl-CoA carboxylase (ACC1), and Diacylglyceride acyl-transferase (DGA1) in Y. lipolytica yielded an engineered strain exhibiting highly desirable phenotypes of fast cell growth and lipid overproduction including high carbon to lipid conversion yield (84.7% of theoretical maximal yield), high lipid titers (~55g/L), enhanced tolerance to glucose and cellulose-derived sugars. Moreover, the engineered strain featured a three-fold growth advantage over the wild type strain. As a result, a maximal lipid productivity of ~1g/L/h is obtained during the stationary phase. Furthermore, we showed that the engineered yeast required cytoskeleton remodeling in eliciting the obesity phenotype. Altogether, our work describes the development of a microbial catalyst with the highest reported lipid yield, titer and productivity to date. This is an important step towards the development of an efficient and cost-effective process for biodiesel production from renewable resources.
Assuntos
Lipídeos , Engenharia Metabólica , Yarrowia , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipídeos/biossíntese , Lipídeos/genética , Estearoil-CoA Dessaturase , Yarrowia/genética , Yarrowia/metabolismoRESUMO
ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1(-/-) mice have reduced viability and fail to thrive early after birth. Male Ube2j1(-/-) mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1(-/-) elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.
Assuntos
Espermatogênese , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Imunoglobulinas/metabolismo , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Espermátides/citologia , Espermátides/patologia , Enzimas de Conjugação de Ubiquitina/deficiência , Resposta a Proteínas não Dobradas , Regulação para CimaRESUMO
Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.
Assuntos
Diferenciação Celular/genética , Fertilidade/genética , Células Germinativas/fisiologia , Sirtuína 1/metabolismo , Espermatogênese/genética , Acetilação , Animais , Diferenciação Celular/fisiologia , Montagem e Desmontagem da Cromatina/genética , Cromatografia Líquida , Feminino , Fertilidade/fisiologia , Imunofluorescência , Histonas/metabolismo , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional/genética , Sirtuína 1/deficiência , Espectrometria de Massas em Tandem , Testículo/metabolismoRESUMO
In man, mutations in different regions of the prion protein (PrP) are associated with infectious neurodegenerative diseases that have remarkably different clinical signs and neuropathological lesions. To explore the roots of this phenomenon, we created a knock-in mouse model carrying the mutation associated with one of these diseases [Creutzfeldt-Jakob disease (CJD)] that was exactly analogous to a previous knock-in model of a different prion disease [fatal familial insomnia (FFI)]. Together with the WT parent, this created an allelic series of three lines, each expressing the same protein with a single amino acid difference, and with all native regulatory elements intact. The previously described FFI mice develop neuronal loss and intense reactive gliosis in the thalamus, as seen in humans with FFI. In contrast, CJD mice had the hallmark features of CJD, spongiosis and proteinase K-resistant PrP aggregates, initially developing in the hippocampus and cerebellum but absent from the thalamus. A molecular transmission barrier protected the mice from any infectious prion agents that might have been present in our mouse facility and allowed us to conclude that the diseases occurred spontaneously. Importantly, both models created agents that caused a transmissible neurodegenerative disease in WT mice. We conclude that single codon differences in a single gene in an otherwise normal genome can cause remarkably different neurodegenerative diseases and are sufficient to create distinct protein-based infectious elements.
Assuntos
Códon/genética , Modelos Animais de Doenças , Mutação , Doenças Priônicas/genética , Príons/genética , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/transmissão , Feminino , Humanos , Imuno-Histoquímica , Insônia Familiar Fatal/genética , Insônia Familiar Fatal/metabolismo , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Fenótipo , Doenças Priônicas/metabolismo , Doenças Priônicas/transmissão , Príons/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Proteínas de Homeodomínio/imunologia , Lectinas Tipo C/imunologia , Macrófagos Peritoneais/imunologia , Neuropeptídeos/imunologia , Fagocitose/imunologia , Proteínas Tirosina Quinases/imunologia , Actinas/genética , Actinas/imunologia , Actinas/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Candida albicans/metabolismo , Candidíase/genética , Candidíase/metabolismo , Candidíase/patologia , Linhagem Celular , Diglicerídeos/genética , Diglicerídeos/imunologia , Diglicerídeos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/imunologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
Protein quality control is a balance between chaperone-assisted folding and removal of misfolded proteins from the endoplasmic reticulum (ER). Cell-based assays have been used to identify key players of the dislocation machinery, including members of the Derlin family. We generated conditional knockout mice to examine the in vivo role of Derlin-2, a component that nucleates cellular dislocation machinery. In most Derlin-2-deficient tissues, we found constitutive upregulation of ER chaperones and IRE-1-mediated induction of the unfolded protein response. The IRE-1/XBP-1 pathway is required for development of highly secretory cells, particularly plasma cells and hepatocytes. However, B lymphocyte development and antibody secretion were normal in the absence of Derlin-2. Likewise, hepatocyte function was unaffected by liver-specific deletion of Derlin-2. Whole-body deletion of Derlin-2 results in perinatal death. The few mice that survived to adulthood all developed skeletal dysplasia, likely caused by defects in collagen matrix protein secretion by costal chondrocytes.
Assuntos
Condrócitos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Transporte Proteico , Resposta a Proteínas não Dobradas , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Condrócitos/citologia , Embrião de Mamíferos/anormalidades , Feminino , Morte Fetal/genética , Fibroblastos/metabolismo , Hepatócitos/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Regulação para CimaRESUMO
Fatty acid transport protein (FATP)2, a member of the FATP family of fatty acid uptake mediators, has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase (VLACS). Here we address whether FATP2 is 1) a peroxisomal enzyme, 2) a plasma membrane-associated long-chain fatty acid (LCFA) transporter, or 3) a multifunctional protein. We found that, in mouse livers, only a minor fraction of FATP2 localizes to peroxisomes, where it contributes to approximately half of the peroxisomal VLACS activity. However, total hepatic (V)LACS activity was not significantly affected by loss of FATP2, while LCFA uptake was reduced by 40%, indicating a more prominent role in hepatic LCFA uptake. This suggests FATP2 as a potential target for a therapeutic intervention of hepatosteatosis. Adeno-associated virus 8-based short hairpin RNA expression vectors were used to achieve liver-specific FATP2 knockdown, which significantly reduced hepatosteatosis in the face of continued high-fat feeding, concomitant with improvements in liver physiology, fasting glucose, and insulin levels. Based on our findings, we propose a model in which FATP2 is a multifunctional protein that shows subcellular localization-dependent activity and is a major contributor to peroxisomal (V)LACS activity and hepatic fatty acid uptake, suggesting FATP2 as a potential novel target for the treatment of nonalcoholic fatty liver disease.
Assuntos
Coenzima A Ligases/metabolismo , Fígado/enzimologia , Peroxissomos/enzimologia , Animais , Transporte Biológico , Western Blotting , Fígado Gorduroso/enzimologia , Inativação Gênica , Hepatócitos/enzimologia , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and AII amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections.
Assuntos
Materiais Biocompatíveis , Decanoatos/metabolismo , Glicerol/análogos & derivados , Polímeros/metabolismo , Retina/transplante , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Células Cultivadas , Técnicas de Cocultura , Glicerol/metabolismo , Humanos , Teste de Materiais , Nanofibras/química , Proteínas de Neurofilamentos/metabolismo , Oligopeptídeos/metabolismo , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Recoverina/metabolismo , Retina/citologia , Retina/metabolismo , Propriedades de Superfície , Suínos , Vimentina/metabolismoRESUMO
A crucial tenet of the prion hypothesis is that misfolding of the prion protein (PrP) induced by mutations associated with familial prion disease is, in an otherwise normal mammalian brain, sufficient to generate the infectious agent. Yet this has never been demonstrated. We engineered knockin mice to express a PrP mutation associated with a distinct human prion disease, fatal familial insomnia (FFI). An additional substitution created a strong transmission barrier against pre-existing prions. The mice spontaneously developed a disease distinct from that of other mouse prion models and highly reminiscent of FFI. Unique pathology was transmitted from FFI mice to mice expressing wild-type PrP sharing the same transmission barrier. FFI mice were highly resistant to infection by pre-existing prions, confirming infectivity did not arise from contaminating agents. Thus, a single amino acid change in PrP is sufficient to induce a distinct neurodegenerative disease and the spontaneous generation of prion infectivity.