RESUMEN

Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.

Asunto(s)

Colágeno , Microscopía por Crioelectrón , Ciclofilinas , Proteínas de la Matriz Extracelular , Humanos , Colágeno/metabolismo , Colágeno/química , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Ciclofilinas/metabolismo , Ciclofilinas/química , Ciclofilinas/genética , Dominio Catalítico , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Procesamiento Proteico-Postraduccional , Sitios de Unión , Unión Proteica , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Modelos Moleculares , Mutación , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/química , Glicoproteínas de Membrana , Proteoglicanos , Chaperonas Moleculares , Prolil Hidroxilasas

RESUMEN

TOR (target of rapamycin), a ubiquitous protein kinase central to cellular homeostasis maintenance, fundamentally regulates ribosome biogenesis in part by its target La-related protein 1 (LARP1). Among other target transcripts, LARP1 specifically binds TOP (terminal oligopyrimidine) mRNAs encoding all 80 ribosomal proteins in a TOR-dependent manner through its C-terminal region containing the DM15 module. Though the functional implications of the LARP1 interaction with target mRNAs is controversial, it is clear that the TOP-LARP1-TOR axis is critical to cellular health in humans. Its existence and role in evolutionarily divergent animals remain less understood. We focused our work on expanding our knowledge of the first arm of the axis: the connection between LARP1-DM15 and the 5' TOP motif. We show that the overall DM15 architecture observed in humans is conserved in fruit fly and zebrafish. Both adopt familiar curved arrangements of HEAT-like repeats that bind 5' TOP mRNAs on the same conserved surface, although molecular dynamics simulations suggest that the N-terminal fold of the fruit fly DM15 is predicted to be unstable and unfold. We demonstrate that each ortholog interacts with TOP sequences with varying affinities. Importantly, we determine that the ability of the DM15 region to bind some TOP sequences but not others might amount to the context of the RNA structure, rather than the ability of the module to recognize some sequences but not others. We propose that TOP mRNAs may retain similar secondary structures to regulate LARP1 DM15 recognition.

Asunto(s)

Autoantígenos , Evolución Molecular , Ribonucleoproteínas , Antígeno SS-B , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/química , Autoantígenos/metabolismo , Autoantígenos/genética , Autoantígenos/química , Animales , Humanos , Pez Cebra/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Unión Proteica

RESUMEN

La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].

Asunto(s)

Poli A , Unión Proteica , Ribonucleoproteínas , Antígeno SS-B , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Poli A/metabolismo , Poli A/química , Humanos , Modelos Moleculares , Sitios de Unión , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Cristalografía por Rayos X , Dominios Proteicos , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/química , ARN Mensajero/metabolismo , ARN Mensajero/química , ARN Mensajero/genética

RESUMEN

The mammalian Golgi is composed of stacks that are laterally connected into a continuous ribbon-like structure. The integrity and function of the ribbon is disrupted under stress conditions, but the molecular mechanisms remain unclear. Here we show that the ribbon is maintained by biomolecular condensates of RNA and the Golgi matrix protein GM130 (GOLGA2). We identify GM130 as a membrane-bound RNA-binding protein, which directly recruits RNA and associated RNA-binding proteins to the Golgi membrane. Acute degradation of RNA or GM130 in cells disrupts the ribbon. Under stress conditions, RNA dissociates from GM130 and the ribbon is disjointed, but after the cells recover from stress the ribbon is restored. When overexpressed in cells, GM130 forms RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its amino terminus, which binds RNA to induce liquid-liquid phase separation. These co-condensates are sufficient to link purified Golgi membranes, reconstructing lateral linking of stacks into a ribbon-like structure. Together, these studies show that RNA acts as a structural biopolymer that together with GM130 maintains the integrity of the Golgi ribbon.

Asunto(s)

Autoantígenos , Aparato de Golgi , Proteínas de la Membrana , ARN , Aparato de Golgi/metabolismo , Humanos , Autoantígenos/metabolismo , Autoantígenos/genética , Autoantígenos/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/química , ARN/metabolismo , ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/química , Células HeLa , Condensados Biomoleculares/metabolismo , Unión Proteica , Membranas Intracelulares/metabolismo , Animales , Células HEK293

RESUMEN

The scavenging ability of cerium oxide nanoparticles (CeNPs) for reactive oxygen species has been intensively studied in the field of catalysis. However, the immunological impact of these particles has not yet been thoroughly investigated, despite intensive research indicating that modulation of the reactive oxygen species could potentially regulate cell fate and adaptive immune responses. In this study, we examined the intrinsic capability of CeNPs to induce tolerogenic dendritic cells via their reactive oxygen species-scavenging effect when the autoantigenic peptides were simply mixed with CeNPs. CeNPs effectively reduced the intracellular reactive oxygen species levels in dendritic cells in vitro, leading to the suppression of costimulatory molecules as well as NLRP3 inflammasome activation, even in the presence of pro-inflammatory stimuli. Subcutaneously administrated PEGylated CeNPs were predominantly taken up by antigen-presenting cells in lymph nodes and to suppress cell maturation in vivo. The administration of a mixture of PEGylated CeNPs and myelin oligodendrocyte glycoprotein peptides, a well-identified autoantigen associated with antimyelin autoimmunity, resulted in the generation of antigen-specific Foxp3+ regulatory T cells in mouse spleens. The induced peripheral regulatory T cells actively inhibited the infiltration of autoreactive T cells and antigen-presenting cells into the central nervous system, ultimately protecting animals from experimental autoimmune encephalomyelitis when tested using a mouse model mimicking human multiple sclerosis. Overall, our findings reveal the potential of CeNPs for generating antigen-specific immune tolerance to prevent multiple sclerosis, opening an avenue to restore immune tolerance against specific antigens by simply mixing the well-identified autoantigens with the immunosuppressive CeNPs.

Asunto(s)

Cerio , Células Dendríticas , Encefalomielitis Autoinmune Experimental , Tolerancia Inmunológica , Nanopartículas , Péptidos , Especies Reactivas de Oxígeno , Cerio/química , Cerio/farmacología , Animales , Especies Reactivas de Oxígeno/metabolismo , Ratones , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Nanopartículas/química , Células Dendríticas/inmunología , Células Dendríticas/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Péptidos/inmunología , Ratones Endogámicos C57BL , Autoantígenos/inmunología , Autoantígenos/química , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Femenino , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología

RESUMEN

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.

Asunto(s)

Secuencias de Aminoácidos , Resonancia Magnética Nuclear Biomolecular , Humanos , Secuencia de Aminoácidos , Autoantígenos/química , Autoantígenos/metabolismo , Isótopos de Nitrógeno , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas de Unión al ARN

RESUMEN

There is no curative treatment for chronic auto-inflammatory diseases including rheumatoid arthritis, and current treatments can induce off-target side effects due to systemic immune suppression. This work has previously shown that dexamethasone-pulsed tolerogenic dendritic cells loaded with the arthritis-specific antigen human proteoglycan can suppress arthritis development in a proteoglycan-induced arthritis mouse model. To circumvent ex vivo dendritic cell culture, and enhance antigen-specific effects, drug delivery vehicles, such as liposomes, provide an interesting approach. Here, this work uses anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol liposomes with enhanced loading of human proteoglycan-dexamethasone conjugates by cationic lysine tetramer addition. Antigen-pulsed tolerogenic dendritic cells induced by liposomal dexamethasone in vitro enhanced antigen-specific regulatory T cells to a similar extent as dexamethasone-induced tolerogenic dendritic cells. In an inflammatory adoptive transfer model, mice injected with antigen-dexamethasone liposomes have significantly higher antigen-specific type 1 regulatory T cells than mice injected with antigen only. The liposomes significantly inhibit the progression of arthritis compared to controls in preventative and therapeutic proteoglycan-induced arthritis mouse models. This coincides with systemic tolerance induction and an increase in IL10 expression in the paws of mice. In conclusion, a single administration of autoantigen and dexamethasone-loaded liposomes seems to be a promising antigen-specific treatment strategy for arthritis in mice.

Asunto(s)

Autoantígenos , Células Dendríticas , Dexametasona , Liposomas , Animales , Liposomas/química , Dexametasona/química , Dexametasona/farmacología , Ratones , Autoantígenos/inmunología , Autoantígenos/química , Células Dendríticas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Humanos , Artritis Experimental/inmunología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/terapia , Proteoglicanos/química , Proteoglicanos/farmacología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Artritis Reumatoide/inmunología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/terapia , Artritis Reumatoide/inducido químicamente

RESUMEN

Centromeric chromatin is thought to play a critical role in ensuring the faithful segregation of chromosomes during mitosis. However, our understanding of this role is presently limited by our poor understanding of the structure and composition of this unique chromatin. The nucleosomal variant, CENP-A, localizes to narrow regions within the centromere, where it plays a major role in centromeric function, effectively serving as a platform on which the kinetochore is assembled. Previous work found that, within a given cell, the number of microtubules within kinetochores is essentially unchanged between CENP-A-localized regions of different physical sizes. However, it is unknown if the amount of CENP-A is also unchanged between these regions of different sizes, which would reflect a strict structural correspondence between these two key characteristics of the centromere/kinetochore assembly. Here, we used super-resolution optical microscopy to image and quantify the amount of CENP-A and DNA within human centromere chromatin. We found that the amount of CENP-A within CENP-A domains of different physical sizes is indeed the same. Further, our measurements suggest that the ratio of CENP-A- to H3-containing nucleosomes within these domains is between 8:1 and 11:1. Thus, our results not only identify an unexpectedly strict relationship between CENP-A and microtubules stoichiometries but also that the CENP-A centromeric domain is almost exclusively composed of CENP-A nucleosomes.

Asunto(s)

Microscopía , Nucleosomas , Humanos , Proteína A Centromérica/genética , Proteínas Cromosómicas no Histona/metabolismo , Centrómero/metabolismo , Cromatina , Cinetocoros/metabolismo , Autoantígenos/química

RESUMEN

Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public ß-chain variable region-complementary-determining region 3ß (BV9-CDR3ß) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3ß TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

Asunto(s)

Autoinmunidad , Antígenos HLA-B , Péptidos , Receptores de Antígenos de Linfocitos T , Humanos , Autoantígenos/química , Autoantígenos/inmunología , Autoantígenos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígenos HLA-B/inmunología , Antígenos HLA-B/metabolismo , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Líquido Sinovial/inmunología , Espondilitis Anquilosante/inmunología , Uveítis Anterior/inmunología , Biblioteca de Péptidos , Reacciones Cruzadas , Secuencias de Aminoácidos

RESUMEN

CENP-A is a histone variant found in high abundance at the centromere in humans. At the centromere, this histone variant replaces the histone H3 found throughout the bulk chromatin. Additionally, the centromere comprises tandem repeats of α-satellite DNA, which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here, we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on α-satellite DNA did not show any positional preference along the substrate, which is similar to both H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on α-satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly.

Asunto(s)

División del Núcleo Celular , Proteína A Centromérica , Centrómero , ADN , Histonas , Nucleosomas , Autoantígenos/química , Autoantígenos/genética , División del Núcleo Celular/genética , División del Núcleo Celular/fisiología , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , ADN Satélite , Histonas/genética , Histonas/metabolismo , Humanos , Microscopía de Fuerza Atómica , Nucleosomas/genética , Nucleosomas/metabolismo

RESUMEN

GW182 family proteins are a key component of microRNA-protein complex eliciting translational repression and/or degradation of microRNA-targets. The microRNAs in complex with Argonaute proteins bind to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4-NOT complex which accelerates deadenylation and inhibits translation. The carboxyl-terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4-NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino-terminal Argonaute-binding domain of human GW182/TNRC6A can associate with the CCR4-NOT complex. This region resides between the two Argonaute-binding sites and contains reiterated GW/WG-motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4-NOT complex. Furthermore, co-expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4-NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4-NOT complex.

Asunto(s)

Proteínas Argonautas , Autoantígenos , MicroARNs , Proteínas de Unión al ARN , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/metabolismo , Sitios de Unión , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores CCR4/genética , Receptores CCR4/metabolismo , Factores de Transcripción/metabolismo , Triptófano/genética , Triptófano/metabolismo

RESUMEN

The leiomodin1 (LMOD1) gene, encoding a potent actin nucleator, was recently reported as a potential pathogenic gene of megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS, OMIM 619362). However, only a single patient has been reported to have LMOD1 mutations, and the underlying pathogenic mechanism remains unknown. Here, we described a male infant with LMOD1 mutations presenting typical symptoms of pediatric intestinal pseudo-obstruction (PIPO) but without megacystis and microcolon. Two compound heterozygous missense variants (c.1106C>T, p.T369M; c.1262G>A, p.R421H) were identified, both affecting highly conserved amino acid residues within the second actin-binding site (ABS2) domain of LMOD1. Expression analysis showed that both variants resulted in significantly reduced protein amounts, especially for p.T369M, which was almost undetectable. The reduction was only partially rescued by the proteasome inhibitor MG-132, indicating that there might be proteasome-independent pathways involved in the degradation of the mutant proteins. Molecular modeling showed that variant p.T369M impaired the local protein conformation of the ABS2 domain, while variant p.R421H directly impaired the intermolecular interaction between ABS2 and actin. Accordingly, both variants significantly damaged LMOD1-mediated actin nucleation. These findings provide further human genetic evidence supporting LMOD1 as a pathogenic gene underlying visceral myopathy including PIPO and MMIHS, strengthen the critical role of ABS2 domain in LMOD1-mediated actin nucleation, and moreover, reveal an unrecognized role of ABS2 in protein stability.

Asunto(s)

Actinas/metabolismo , Autoantígenos/genética , Proteínas del Citoesqueleto/genética , Seudoobstrucción Intestinal/genética , Mutación con Pérdida de Función , Autoantígenos/química , Autoantígenos/metabolismo , Sitios de Unión , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Células HeLa , Humanos , Lactante , Seudoobstrucción Intestinal/metabolismo , Seudoobstrucción Intestinal/patología , Masculino , Simulación del Acoplamiento Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estabilidad Proteica

RESUMEN

Like other human oncoproteins, Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) exerts cancer promoting function through interaction with other partner proteins, such as MYC and Protein Phosphatase 2A (PP2A). CIP2A regulates several MYC-independent and/or dependent gene expression programs. Broadly, CIP2A can inhibit PP2A, and especially it has been shown to inhibit MYC-associated PP2A, precisely to increase MYC stability and function. Availability of crystal structure has broached the research focus to develop new therapeutics targeting CIP2A. In the present study, structural information of the protein has been used for identification of modulators for homo-dimer CIP2A using advanced structure-based drug design approaches. The compound library, 'Maybridge Screening Collection' database (∼62,000 compounds) has been virtually screened to find out potent modulators for CIP2A. Identification of hotspot region on CIP2A protein-protein interaction interface has been performed using three different tools (HotPoint, SiteMap and icmPocketFinder). Thereafter, molecular docking (Extra Precision and Induced Fit Docking), and long range molecular dynamics simulation analysis, and ADME profile analysis have been carried out for screening purposes. Calculations of MM-PBSA based binding free energy (ΔG), and Density Functional Theory for quantum chemical simulations have been carried out for the hit compounds obtained through multi-step molecular docking based virtual screening technique. The multi-chemometric studies suggested that hit modulators have formed significant numbers of molecular interactions with hotspot residues in the homo-dimer interface region, which enable to hold CIP2A binding stability. Compounds with average ΔG values (ranging -3.4 x 10-2 to -1.1 x 10-2 KJ/mol) signifying promising CIP2A modulators.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Autoantígenos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Autoantígenos/química , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Simulación del Acoplamiento Molecular , Proteína Fosfatasa 2

RESUMEN

BACKGROUND: Bullous pemphigoid (BP) mostly involves elderly patients. The diagnosis of BP requires special immunological tests, which makes some patients unable to be diagnosed and treated timely. OBJECTIVE: The accuracy and application value of immune colloidal gold technique (ICGT) in BP were evaluated. The colloidal gold was conjugated with recombinant BP180 NC16A protein and mouse IgG antibody. As the test and control lines, the mouse-anti-human IgG and goat-anti-mouse IgG, respectively, were blotted on the nitrocellulose membrane. METHODS: 414 serum samples of consecutive patients with suspected BP and 15 samples from healthy donors were recruited. The consistency between ICGT and ELISA, and between serum and plasma/whole blood were evaluated. Subgroup analyses were performed in terms of clinical characteristics. We also followed up 65 BP patients' strip results to explore the predictive value of ICGT. RESULTS: Strong agreements between ICGT and ELISA(κ = 0.902) and between plasma/whole blood and serum samples (κ = 0.980) with good stability were observed. The ICGT achieved sensitivity of 93.9%, and specificity of 97.6%. In subgroup analysis, the sensitivity was significantly higher in older patients (96.3%), and with more typical lesions such as blisters (96.2%) and erosions (92.4%). In follow-up, we also found BP patients who kept ICGT-negative in remission state all got consecutive positive strips 1-3 weeks prior to mild new activity or flare. CONCLUSION: ICGT shows high potential as a rapid and stable option for the diagnosis and monitoring of BP. Further investigations are needed to re-evaluate this technique in a prospective study with a multicenter design.

Asunto(s)

Autoantígenos , Colágenos no Fibrilares , Penfigoide Ampolloso , Humanos , Autoanticuerpos , Autoantígenos/química , Autoantígenos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G , Colágenos no Fibrilares/química , Colágenos no Fibrilares/inmunología , Penfigoide Ampolloso/diagnóstico por imagen , Estudios Prospectivos , Oro Coloide/química , Colágeno Tipo XVII

RESUMEN

Excessive synthesis of type I collagen is a hallmark of fibrotic diseases. Binding of La-related protein 6 (LARP6) to the 5' stem-loop (5'SL) of collagen mRNAs regulates their translation leading to an unnaturally elevated rate of collagen biosynthesis in fibrosis. Previous work suggested that LARP6 needs two domains to form stable complex with 5'SL RNA, the La domain and the juxtaposed RNA recognition motif (RRM), jointly called the La-module. Here we describe that La domain of LARP6 is necessary and sufficient for recognition of 5'SL in RNA sequence specific manner. A three-amino-acid motif located in the flexible loop connecting the second α-helix to the ß-sheet of the La domain, called the RNK-motif, is critical for binding. Mutation of any of these three amino acids abolishes the binding of the La domain to 5'SL. The major site of crosslinking of LARP6 to 5'SL RNA was mapped to this motif, as well. The RNK-motif is not found in other LARPs, which cannot bind 5'SL. Presence of RRM increases the stability of complex between La domain and 5'SL RNA and RRM domain does not make extensive contacts with 5'SL RNA. We propose a model in which the initial recognition of 5'SL by LARP6 is mediated by the RNK epitope and further stabilized by the RRM domain. This discovery suggests that the interaction between LARP6 and collagen mRNAs can be blocked by small molecules that target the RNK epitope and will help rational design of the LARP6 binding inhibitors as specific antifibrotic drugs.

Asunto(s)

Autoantígenos/química , Colágeno Tipo I/química , Fibrosis/metabolismo , ARN Mensajero/química , Ribonucleoproteínas/química , Secuencias de Aminoácidos , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno , Colágeno Tipo I/biosíntesis , Humanos , Conformación de Ácido Nucleico , Preparaciones Farmacéuticas , Unión Proteica , Dominios Proteicos , ARN Mensajero/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Antígeno SS-B

RESUMEN

This study aimed to establish a cell-based assay (CBA) for the detection of agrin antibodies (Agrin-Ab) to explore the clinical features of agrin antibody-positive Chinese patients with myasthenia gravis (Agrin-MG). We developed a CBA based on the human full-length agrin protein expressed in HEK293T cells for the reliable and efficient detection of Agrin-Ab. Clinical data and serum samples were collected from 1948 MG patients in 26 provinces in China. The demographic and clinical features of Agrin-MG patients were compared with those of other MG patient subsets. Eighteen Agrin-MG cases were identified from 1948 MG patients. Nine patients were Agrin-Ab positive, and nine were AChR-Ab and Agrin-Ab double-positive (Agrin/AChR-MG). Eleven (61.11%) patients were males older than 40 years of age. The initial symptom in 13 (81.25%) cases was ocular weakness. Occasionally, the initial symptom was limb-girdle weakness (two cases) or bulbar muscle weakness (one case). Agrin-MG patients demonstrated slight improvement following treatment with either acetylcholinesterase inhibitor or prednisone; however, the combination of the two drugs could effectively relieve MG symptoms. In China, Agrin-MG demonstrated seropositivity rates of 0.92%. These patients were commonly middle-aged or elderly men. The patients usually presented weakness in the ocular, bulbar, and limb muscles, which may be combined with thymoma. These patients have more severe diseases, although the combination of pyridostigmine and prednisone was usually effective in relieving symptoms.

Asunto(s)

Agrina/inmunología , Autoanticuerpos/sangre , Autoantígenos/inmunología , Miastenia Gravis/inmunología , Prednisona , Edad de Inicio , Anciano , Agrina/química , Agrina/genética , Autoantígenos/química , Autoantígenos/genética , China/epidemiología , Inhibidores de la Colinesterasa/uso terapéutico , Femenino , Geografía Médica , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Debilidad Muscular/etiología , Miastenia Gravis/etnología , Miastenia Gravis/etiología , Prednisona/uso terapéutico , Proteínas Recombinantes/inmunología , Timoma/complicaciones , Neoplasias del Timo/complicaciones

RESUMEN

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.

Asunto(s)

Pliegue del ARN , ARN Largo no Codificante/química , Proteínas de Unión al ARN/metabolismo , Inactivación del Cromosoma X , Animales , Autoantígenos/química , Autoantígenos/metabolismo , Sitios de Unión , Línea Celular , Ratones , Unión Proteica , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética

RESUMEN

Antibodies against FGFR3 define a subgroup of sensory neuropathy (SN). The aim of this study was to identify the epitope(s) of anti-FGFR3 autoantibodies and potential epitope-dependent clinical subtypes. Using SPOT methodology, five specific candidate epitopes, three in the juxtamembrane domain (JMD) and two in the tyrosine kinase domain (TKD), were screened with 68 anti-FGFR3-positive patients and 35 healthy controls. The identified epitopes cover 6/15 functionally relevant sites of the protein. Four patients reacted with the JMD and 11 with the TKD, partly even in a phosphorylation-state dependent manner. The epitope could not be identified in the others. Patients with antibodies recognizing TKD exhibited a more severe clinical and electrophysiological impairment than others.

Asunto(s)

Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Epítopos/inmunología , Proteínas del Tejido Nervioso/inmunología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/inmunología , Trastornos de la Sensación/inmunología , Adulto , Autoanticuerpos/sangre , Autoantígenos/química , Femenino , Ganglios Espinales/inmunología , Humanos , Masculino , Persona de Mediana Edad , Fosforilación , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/química , Células Receptoras Sensoriales/inmunología

RESUMEN

Decades ago, we and many other groups showed a nucleo-cytoplasmic translocation of La protein in cultured cells. This shuttling of La protein was seen after UV irradiation, virus infections, hydrogen peroxide exposure and the Fenton reaction based on iron or copper ions. All of these conditions are somehow related to oxidative stress. Unfortunately, these harsh conditions could also cause an artificial release of La protein. Even until today, the shuttling and the cytoplasmic function of La/SS-B is controversially discussed. Moreover, the driving mechanism for the shuttling of La protein remains unclear. Recently, we showed that La protein undergoes redox-dependent conformational changes. Moreover, we developed anti-La monoclonal antibodies (anti-La mAbs), which are specific for either the reduced form of La protein or the oxidized form. Using these tools, here we show that redox-dependent conformational changes are the driving force for the shuttling of La protein. Moreover, we show that translocation of La protein to the cytoplasm can be triggered in a ligand/receptor-dependent manner under physiological conditions. We show that ligands of toll-like receptors lead to a redox-dependent shuttling of La protein. The shuttling of La protein depends on the redox status of the respective cell type. Endothelial cells are usually resistant to the shuttling of La protein, while dendritic cells are highly sensitive. However, the deprivation of intracellular reducing agents in endothelial cells makes endothelial cells sensitive to a redox-dependent shuttling of La protein.

Asunto(s)

Transporte Activo de Núcleo Celular , Autoantígenos/química , Núcleo Celular/metabolismo , Oxígeno/química , Ribonucleoproteínas/química , Anticuerpos Monoclonales/química , Citoplasma/metabolismo , Epítopos/química , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Óxido Nítrico/metabolismo , Oxidación-Reducción , Conformación Proteica , Transducción de Señal , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Rayos Ultravioleta , Antígeno SS-B

RESUMEN

Background: Acute rheumatic fever (ARF) is a serious sequela of Group A Streptococcus (GAS) infection associated with significant global mortality. Pathogenesis remains poorly understood, with the current prevailing hypothesis based on molecular mimicry and the notion that antibodies generated in response to GAS infection cross-react with cardiac proteins such as myosin. Contemporary investigations of the broader autoantibody response in ARF are needed to both inform pathogenesis models and identify new biomarkers for the disease. Methods: This study has utilised a multi-platform approach to profile circulating autoantibodies in ARF. Sera from patients with ARF, matched healthy controls and patients with uncomplicated GAS pharyngitis were initially analysed for autoreactivity using high content protein arrays (Protoarray, 9000 autoantigens), and further explored using a second protein array platform (HuProt Array, 16,000 autoantigens) and 2-D gel electrophoresis of heart tissue combined with mass spectrometry. Selected autoantigens were orthogonally validated using conventional immunoassays with sera from an ARF case-control study (n=79 cases and n=89 matched healthy controls) and a related study of GAS pharyngitis (n=39) conducted in New Zealand. Results: Global analysis of the protein array data showed an increase in total autoantigen reactivity in ARF patients compared with controls, as well as marked heterogeneity in the autoantibody profiles between ARF patients. Autoantigens previously implicated in ARF pathogenesis, such as myosin and collagens were detected, as were novel candidates. Disease pathway analysis revealed several autoantigens within pathways linked to arthritic and myocardial disease. Orthogonal validation of three novel autoantigens (PTPN2, DMD and ANXA6) showed significant elevation of serum antibodies in ARF (p < 0.05), and further highlighted heterogeneity with patients reactive to different combinations of the three antigens. Conclusions: The broad yet heterogenous elevation of autoantibodies observed suggests epitope spreading, and an expansion of the autoantibody repertoire, likely plays a key role in ARF pathogenesis and disease progression. Multiple autoantigens may be needed as diagnostic biomarkers to capture this heterogeneity.

Asunto(s)

Autoanticuerpos/sangre , Autoantígenos/química , Análisis por Matrices de Proteínas , Fiebre Reumática/sangre , Streptococcus pyogenes , Niño , Humanos , Nueva Zelanda