RESUMO
The epithelial lining of the small intestine consists of multiple cell types, including Paneth cells and goblet cells, that work in cohort to maintain gut health. 3D in vitro cultures of human primary epithelial cells, called organoids, have become a key model to study the functions of Paneth cells and goblet cells in normal and diseased conditions. Advances in these models include the ability to skew differentiation to particular lineages, providing a useful tool to study cell type specific function/dysfunction in the context of the epithelium. Here, we use comprehensive profiling of mRNA, microRNA and long non-coding RNA expression to confirm that Paneth cell and goblet cell enrichment of murine small intestinal organoids (enteroids) establishes a physiologically accurate model. We employ network analysis to infer the regulatory landscape altered by skewing differentiation, and using knowledge of cell type specific markers, we predict key regulators of cell type specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1b and Myc specific for goblet cells and Ets1, Nr3c1 and Vdr shared between them. Links identified between these regulators and cellular phenotypes of inflammatory bowel disease (IBD) suggest that global regulatory rewiring during or after differentiation of Paneth cells and goblet cells could contribute to IBD aetiology. Future application of cell type enriched enteroids combined with the presented computational workflow can be used to disentangle multifactorial mechanisms of these cell types and propose regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn's disease or ulcerative colitis.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Células Caliciformes/metabolismo , Intestino Delgado/metabolismo , Organoides/metabolismo , Celulas de Paneth/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Feminino , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Masculino , Camundongos Endogâmicos C57BL , Organoides/citologiaRESUMO
The large cytoplasmic DNA viruses such as poxviruses, iridoviruses, and African swine fever virus (ASFV) assemble in discrete perinuclear foci called viral factories. Factories exclude host proteins, suggesting that they are novel subcellular structures induced by viruses. Novel perinuclear structures, called aggresomes are also formed by cells in response to misfolded protein (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883--1898; García-Mata, R., Z. Bebök, E.J. Sorscher, and E.S. Sztul. 1999. J. Cell Biol. 146:1239--1254). In this study, we have investigated whether aggresomes and viral factories are related structures. Aggresomes were compared with viral factories produced by ASFV. Aggresomes and viral factories were located close to the microtubule organizing center and required an intact microtubular network for assembly. Both structures caused rearrangement of intermediate filaments and the collapse of vimentin into characteristic cages, and both recruited mitochondria and cellular chaperones. Given that ASFV factories resemble aggresomes, it is possible that a cellular response originally designed to reduce the toxicity of misfolded proteins is exploited by cytoplasmic DNA viruses to concentrate structural proteins at virus assembly sites.
Assuntos
Vírus da Febre Suína Africana/crescimento & desenvolvimento , Organelas/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Organelas/ultraestrutura , Células Vero , Vimentina/isolamento & purificaçãoRESUMO
The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Acetilglucosaminidase , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/isolamento & purificação , Complexo CD3 , Linhagem Celular , Imunofluorescência , Vetores Genéticos , Complexo de Golgi/imunologia , Humanos , Cinética , Lasers , Substâncias Macromoleculares , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/isolamento & purificação , TransfecçãoRESUMO
The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.
Assuntos
Retículo Endoplasmático/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T/biossíntese , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Membrana Celular/imunologia , Membrana Celular/metabolismo , Clonagem Molecular , Cricetinae , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
gammadelta T cells recognise different types of antigen in alternative ways to alphabeta T cells, and thus appear to play a complementary role in the immune response. However, unlike alphabeta T cells, the role or function of gammadelta T cells is still unclear. As pigs possess a high proportion of circulating gammadelta T cells, they are suitable large animal model to study gammadelta T cell functions. This as yet has not been fully exploited, leaving porcine gammadelta T cell biology and its role in immunity in its infancy. Foot-and-mouth disease (FMD) high potency "emergency" vaccines are able to induce early protection from challenge and it has been suggested that, in part, there is some involvement of innate immune responses. The antigen component of the vaccine is able to stimulate purified naive pig gammadelta T cells and induce the mRNA of various cytokines and chemokines. This observation suggests that gammadelta T cells probably contribute to the early phase of the immune responses to FMD vaccination, and perhaps infection. A subset of these circulating gammadelta T cells display a phenotype similar to professional antigen presenting cells and are able to take up and present soluble antigen to CD4(+) T cells in a direct cell-cell interaction via MHC class II. This direct interaction between gammadelta T cells and CD4(+) T cells is likely to have a significant influence on the out come of the adaptive immune response.
Assuntos
Doenças dos Suínos/imunologia , Suínos/imunologia , Subpopulações de Linfócitos T/imunologia , Viroses/veterinária , Adaptação Fisiológica , Animais , Quimiocinas/genética , Citocinas/genética , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Imunidade Inata , Cooperação Linfocítica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Vacinas Virais/imunologia , Vacinas Virais/farmacologia , Viroses/imunologiaRESUMO
The T-cell antigen receptor (alpha beta or gamma delta TCR) is known to associate with four polypeptides (CD3 gamma, delta, epsilon and zeta) to form the TCR-CD3 complex. Although the six chains are well characterized, the molecular mass of the TCR-CD3 complex and stoichiometry of the components are currently uncertain. We analysed the TCR of a T-T hybridoma which expresses two distinct heterodimers. When the hybridoma was incubated with a mAb (MR9.2) specific for the V alpha 10V beta 5.1 heterodimer, both of the heterodimers were lost from the cell surface, as measured with mAb MR9.2 and MR9.7 (V alpha 1V beta 1-specific). The ability to co-modulate V alpha 1V beta 1 and V alpha 10V beta 5.1 suggested that TCR complexes could contain two alpha beta-heterodimers. Density gradient sedimentation analysis provided further evidence for higher order TCR. The sedimentation patterns of the TCR were compared to that of the B-cell antigen receptor and the well-characterized VSV membrane G-protein as well as to soluble marker proteins. Maximal cell surface murine and human TCR sedimentation coefficients were substantially greater than the 9-10S predicted for a 210 kDa monovalent alpha beta gamma delta epsilon 2 zeta 2 structure. The TCR sedimented in mild non-ionic detergents as large 18 +/- 3S complexes co-migrating with a 443 kDa marker protein. In contrast, the IgM B-cell antigen receptor had a maximal sedimentation coefficient of 10 +/- 3S, consistent with a predicted size of approximately 300 kDa. Taken together, the results suggested that T-cell antigen receptors can contain more than one alpha beta-heterodimer which could be incorporated into a minimal divalent 10-chain TCR-CD3 complex (e.g. alpha beta gamma epsilon epsilon delta zeta zeta alpha beta).
Assuntos
Complexo CD3/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Complexo CD3/química , Linhagem Celular , Centrifugação com Gradiente de Concentração , Humanos , Camundongos , Agregação de Receptores , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/químicaRESUMO
A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.
Assuntos
Células Apresentadoras de Antígenos/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Suínos/imunologia , Subpopulações de Linfócitos T/fisiologia , Animais , Ativação Linfocitária , Ovalbumina/imunologiaRESUMO
Oxidized dextrans of increasing molecular weight were bound covalently to Erwinia carotovora asparaginase. The resulting conjugates retained 50% of their enzyme activity and showed marked resistance to proteolysis by trypsin and chymotrypsin and inactivation by asparaginase-specific antibody. When tested in-vivo, the larger molecular weight conjugates showed prolonged circulatory survival in both immune and non-immune animals and failed to elicit full type III hypersensitivity or anaphylactic reactions when injected into sensitized guinea-pigs. Rabbits could tolerate multiple doses of the asparaginase conjugate without developing an immunity to the enzyme. A conjugate showing increased circulatory half-life and lowered antigen reactivity should have therapeutic potential.
Assuntos
Asparaginase/administração & dosagem , Animais , Antígenos , Asparaginase/sangue , Asparaginase/imunologia , Química Farmacêutica , Cromatografia em Gel , Dextranos , Hipersensibilidade a Drogas/etiologia , Estabilidade de Medicamentos , Feminino , Cobaias , Técnicas Imunológicas , Cinética , Masculino , Peso Molecular , Peptídeo Hidrolases , Coelhos , SolubilidadeRESUMO
We describe a method for high-pressure freezing and rapid freeze-substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed 'face to face' without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice-crystal artefact. Freeze-substitution at -90 degrees C for 60 min in acetone containing 2% uranyl acetate, followed by warming to -50 degrees C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.
Assuntos
Substituição ao Congelamento/métodos , Animais , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Congelamento , Pressão Hidrostática , Imuno-Histoquímica , Microscopia Imunoeletrônica , Compostos Organometálicos , Células VeroRESUMO
African swine fever virus (ASFV) is a large enveloped DNA virus that shares the striking icosahedral symmetry of iridoviruses. To understand the mechanism of assembly of ASFV, we have been studying the biosynthesis and subcellular distribution of p73, the major structural protein of ASFV. Sucrose density sedimentation of lysates prepared from infected cells showed that newly synthesized p73 was incorporated into a complex with a size of 150 to 250 kDa. p73 synthesized by in vitro translation migrated at 70 kDa, suggesting that cellular and/or viral proteins are required for the formation of the 150- to 250-kDa complex. During a 2-h chase, approximately 50% of the newly synthesized pool of p73 bound to the endoplasmic reticulum (ER). During this period, the membrane-bound pool of p73, but not the cytosolic pool, formed large complexes of approximately 50,000 kDa. The complexes were formed via assembly intermediates, and the entire membrane-associated pool of p73 was incorporated into the 50,000-kDa complex within 2 h. The 50,000-kDa complexes containing p73 were also detected in virions secreted from cells. Immunoprecipitation of sucrose gradients with sera taken from hyperimmune pigs suggested that p73 was the major component of the 50,000-kDa complex. It is possible, therefore, that the complex contains between 600 and 700 copies of p73. The kinetics of complex formation and envelopment of p73 were similar, and complex formation and envelopment were both reversibly inhibited by cycloheximide, suggesting a functional link between complex assembly and ASFV envelopment. A protease protection assay detected 50,000-kDa complexes on the inside and outside of the membranes forming the viral envelope. The identification of a complex containing p73 beneath the envelope of ASFV suggests that p73 may be a component of the inner core shell or matrix of ASFV. The outer pool may represent p73 within the outer capsid layer of the virus. In summary, the data suggest that the assembly of the inner core matrix and outer capsid of ASFV takes place on the ER membrane during envelopment and that these structures are not preassembled in the cytosol.
Assuntos
Vírus da Febre Suína Africana/metabolismo , Retículo Endoplasmático/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Capsídeo/química , Capsídeo/metabolismo , Chlorocebus aethiops , Precursores de Proteínas/química , Células Vero , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Proteínas Estruturais Virais/químicaRESUMO
The T cell receptor for antigen (TCR) is responsible for the recognition of antigen associated with the major histocompatibility complex (MHC). The TCR expressed on the surface of T cells is associated with an invariant structure, CD3. CD3 is assumed to be responsible for intracellular signaling following occupancy of the TCR by ligand. The TCR/CD3 complex consists of six different polypeptides, and represents a uniquely complex multisubunit assembly problem for the cell. The cell copes with this problem by regulating the intracellular assembly of the complex. Within the endoplasmic reticulum, the newly-synthesised chains assemble into the complete structure prior to transport to the cell surface. There are a series of different isoforms of the receptor involving differential use of the TCR heterodimer (alpha-beta or gamma-delta), zeta-family member, and CD3 gamma or delta chains. These are presumably linked to different TCR functions. Assembly of the TCR/CD3 complex competes with specific degradation of unassembled polypeptides. The fate of the receptor depends on the presence of subtle signals on individual chains which determine pairing and assembly or degradation. The T cell is thus able to select a completely assembled fully functional series of distinct TCR/CD3 complexes for expression at the cell surface.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos T/ultraestrutura , Transporte Biológico , Complexo CD3 , Compartimento Celular , Membrana Celular/metabolismo , Chaperoninas , Regulação para Baixo , Genes , Humanos , Lisossomos/metabolismo , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas/fisiologia , Receptores de Antígenos de Linfócitos T/ultraestrutura , Relação Estrutura-AtividadeRESUMO
The T cell antigen receptor is a multiple subunit membrane protein made from six different polypeptide chains (alpha beta gamma delta epsilon zeta). The subunits are transmembrane proteins but only receptors assembled from all six chains are transported efficiently to the plasma membrane. Partial receptors and single subunits fail to reach the Golgi apparatus. This study has used transfected fibroblasts to follow the intracellular fate of the TCR beta and CD3 delta subunits in detail. After a lag period of approximately 90 min both chains were degraded by a process which did not require their transport to the medial Golgi. Degradation was inhibited at temperatures below 18 degrees but was unaffected by agents that disrupted the ER to Golgi transport. Experiments using transfected Chinese hamster ovary Lec 1 cells suggested that CD3 delta was degraded without transport to the cis-Golgi. Lysosomotropic agents had no effect on the proteolysis of the beta chain but did prevent the degradation of approximately 25% of the delta subunit. In the presence of chloroquine the delta subunit could be detected in lysosomes. The experiments show that proteolysis in or close to the endoplasmic reticulum plays a major role in preventing the surface expression of single beta and delta subunits of the T cell antigen receptor; nevertheless, some of the delta chain is able to evade this process. Interestingly, there is a second check on the transport of delta to the plasma membrane, and the subunit is removed from the secretory pathway and delivered to lysosomes for degradation.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Transporte Biológico Ativo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Ensaio de Imunoadsorção Enzimática , Complexo de Golgi/imunologia , Complexo de Golgi/metabolismo , Lisossomos/imunologia , Lisossomos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos T gama-delta , Temperatura , TransfecçãoRESUMO
During endocytosis, mannosylated ligands enter vesicles which have a density intermediate between that of the plasma membrane and secondary lysosomes. Mannosylated ligands are transferred from these vesicles to lysosomes. A solubilization-precipitation assay was used to study the dissociation of mannosylated ligands from their receptor. In whole cells dissociation was rapid (t 1/2 (37 degrees C) = 8 min) and took place before delivery of the ligand to lysosomes. Receptor-ligand dissociation within membrane vesicles, washed free of cytosol, could be induced by addition of ATP and GTP but not ADP. Receptor-ligand dissociation caused by manipulating the pH of the vesicles suggested that the pH within endosomes was lowered to 5.5 by addition of ATP. Dissociation was blocked by proton ionophores and Zn2+, but was unaffected by inhibitors of the F1, Fo-ATPase or the Na+,K+-ATPase. Dissociation did not require Na+ or K+ and was blocked by anion transport inhibitors. Dissociation was slowed in the absence of permeant anions (Cl-). Receptor-ligand complexes within vesicles isolated as early as 2 min following ligand internalization responded to addition of ATP. The results suggest that receptor-ligand dissociation in endosomes requires ATP, possibly to power endosomal acidification via an ATP-dependent proton pump. Dissociation is enhanced in the presence of permeant anions, suggesting the involvement of an anion channel or carrier.
Assuntos
Trifosfato de Adenosina/metabolismo , Macrófagos/metabolismo , Manose/metabolismo , Soroalbumina Bovina/metabolismo , Albumina Sérica , Difosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Fracionamento Celular , Endocitose , Guanosina Trifosfato/metabolismo , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo , Cinética , Ligantes , Potássio/farmacologia , Coelhos , Sódio/farmacologia , Frações Subcelulares/metabolismoRESUMO
It is generally believed that cellular chaperones facilitate the folding of virus capsid proteins, or that capsid proteins fold spontaneously. Here we show that p73, the major capsid protein of African swine fever virus (ASFV) failed to fold and aggregated when expressed alone in cells. This demonstrated that cellular chaperones were unable to aid the folding of p73 and suggested that ASFV may encode a chaperone. An 80-kDa protein encoded by ASFV, termed the capsid-associated protein (CAP) 80, bound to the newly synthesized capsid protein in infected cells. The 80-kDa protein was released following conformational maturation of p73 and dissociated before capsid assembly. Coexpression of the 80-kDa protein with p73 prevented aggregation and allowed the capsid protein to fold with kinetics identical to those seen in infected cells. CAP80 is, therefore, a virally encoded chaperone that facilitates capsid protein folding by masking domains exposed by the newly synthesized capsid protein, which are susceptible to aggregation, but cannot be accommodated by host chaperones. It is likely that these domains are ultimately buried when newly synthesized capsid proteins are added to the growing capsid shell.
Assuntos
Vírus da Febre Suína Africana/química , Capsídeo/química , Chaperonas Moleculares/química , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Sequência de Aminoácidos , Animais , Capsídeo/genética , Regulação Viral da Expressão Gênica , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Dobramento de Proteína , Replicação Viral/genéticaRESUMO
Mannose-lactoperoxidase, a neoglycoprotein prepared by reaction of lactoperoxidase with cyanomethyl 1-thiomannoside, bound to alveolar macrophages at 4 degrees C (Kd = 5.8 X 10(-8) M) and was rapidly internalized at 37 degrees C (K uptake = 2 X 10(-8) M). Mannose-lactoperoxidase binding and uptake were blocked by yeast mannan, and mannose-lactoperoxidase inhibited uptake of 125I-labeled mannose-BSA (bovine serum albumin). Radioiodination of cells with surface-bound mannose-lactoperoxidase was carried out in the presence of glucose and glucose oxidase. A major polypeptide (175 kDa) was radioiodinated by this procedure. Iodination of the 175-kDa polypeptide appeared to be receptor-mediated, since it was blocked by the presence of yeast mannan. Specific iodination was absent from receptor-negative cells. To demonstrate that the 175-kDa species is a ligand-binding protein, cells were iodinated by the standard lactoperoxidase method. Washed cells were then allowed to bind mannose-BSA. Receptor-ligand complexes, prepared by detergent extraction, were passed over anti-BSA IgG affinity columns. Mannose, but not mannose 6-phosphate or galactose, eluted a radioactive protein from the column that migrated with an apparent molecular mass of 175 kDa on NaDodSO4/PAGE. Detergent extracts of crude membranes prepared from macrophage-enriched whole rabbit lung were adsorbed to mannose-Sepharose; the fraction obtained by elution with mannose contained two protein components of 175 and 55 kDa. Subsequent chromatography on N-acetylglucosamine-agarose yielded a single protein of 175 kDa. The 175-kDa polypeptide was shown to bind 125I-labeled mannose-BSA in a precipitation assay. This binding could be blocked with mannan or mannose-BSA. The results indicate that the cell-surface mannose receptor is a 175-kDa protein.
Assuntos
Glicoproteínas/metabolismo , Lectinas Tipo C , Macrófagos/metabolismo , Lectinas de Ligação a Manose , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Receptores Imunológicos/isolamento & purificação , Marcadores de Afinidade , Animais , Membrana Celular/análise , Membrana Celular/metabolismo , Endocitose , Radioisótopos do Iodo , Lactoperoxidase , Macrófagos/análise , Receptor de Manose , Proteínas de Membrana/isolamento & purificação , Peso Molecular , Coelhos , Receptores Imunológicos/metabolismoRESUMO
Recent studies show that the endoplasmic reticulum (ER) contains proteases, but it is not understood how these enzymes are regulated. In this report we study the selective ER degradation of the subunits (alpha beta gamma delta epsilon zeta) of the T-cell antigen receptor (TCR). When analyzed in vivo, unassembled subunits of the TCR fail to reach the Golgi apparatus and show a differential sensitivity to degradation after synthesis. The alpha, beta, and delta subunits are degraded rapidly, while gamma, epsilon, and zeta are stable. To study the regulation of proteolysis in more detail, beta, gamma, delta, and epsilon subunits were expressed alone in fibroblasts and their selective degradation analyzed in vitro. The beta and delta chains were degraded in the complete absence of vesicular transport, indicating their degradation in the ER membrane compartment. Proteolysis was unaffected by GTP gamma S (guanosine 5'-O-(thiotriphosphate)), EDTA, or depletion of ATP. The gamma and epsilon subunits were stable under the same in vitro conditions, indicating that the assay reconstituted selective protein degradation within the ER. Furthermore, the results showed that the gamma and epsilon subunits did not escape degradation by being transported from the ER to pre-Golgi, or cis-Golgi, membrane compartments. Structural determinants of ER degradation contained within the membrane anchor of the TCR beta subunit were only active in permeabilized cells when reducing agents were added to the assay. Surprisingly, reducing conditions disrupted the regulation of ER proteolysis and induced rapid ER degradation of the stable CD3 gamma subunit and of a control interleukin 2 receptor chimera. Taken together, the results indicated that the ER membrane compartment regulates the selective degradation of newly synthesized proteins. Importantly, the stability of proteins retained in the ER was highly sensitive to redox conditions. It is possible that the redox buffer within the ER lumen may regulate ER protein degradation in vivo.
Assuntos
Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Antígenos CD/metabolismo , Células CHO , Cricetinae , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Glutationa/farmacologia , Cinética , Dados de Sequência Molecular , Oligopeptídeos/imunologia , Oxirredução , Coelhos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , TransfecçãoRESUMO
Rabbit alveolar macrophages express a plasma-membrane receptor that recognizes glycoprotein ligands bearing terminal mannose, fucose or N-acetylglucosamine residues. Macrophage membranes were washed extensively with buffers containing high salt and mannose or EDTA to remove endogenously bound ligand, before Triton X-100 extraction. The extracts were chromatographed on mannose-Sepharose. Elution with mannose, followed by dialysis and a second mannose-Sepharose step with EDTA elution, produced a preparation that migrated as single protein band of Mr 175,000 on SDS/polyacrylamide-gel electrophoresis. The purified protein binds mannose-BSA (bovine serum albumin) with a dissociation constant of 1.9 X 10(-8) M. Ligand binding is Ca2+ and pH-dependent, with maximal binding at neutral pH and low binding below pH 6.0. The binding of 125I-mannose-BSA is inhibited by ligands bearing high-mannose oligosaccharides, such as mannan or beta-glucuronidase, as well as the monosaccharides mannose, fucose and N-acetylglucosamine. Galactose, galactosylated BSA, glucose and mannose 6-phosphate are non-inhibitory. Amino acid compositional analyses indicate that the receptor contains high concentrations of aspartate/asparagine and glutamate/glutamine, and low amounts of methionine. The carbohydrate composition was studied by lectin overlays of electrophoretically transferred receptor, and the results indicate the presence of N-linked complex and O-linked sialylated oligosaccharides. A protein of Mr 175,000 was immunoprecipitated from radio-iodinated macrophage membranes with an antibody generated against purified rabbit lung mannose receptor.
Assuntos
Endocitose , Lectinas Tipo C , Macrófagos/metabolismo , Lectinas de Ligação a Manose , Receptores de Superfície Celular , Receptores Imunológicos/metabolismo , Albumina Sérica , Aminoácidos/análise , Animais , Cálcio/farmacologia , Carboidratos/análise , Membrana Celular/metabolismo , Precipitação Química , Cromatografia de Afinidade , Ligantes , Manose/metabolismo , Receptor de Manose , Coelhos , Receptores Imunológicos/efeitos dos fármacos , Receptores Imunológicos/isolamento & purificação , Soroalbumina Bovina/metabolismoRESUMO
The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.