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

Alport syndrome is a hereditary kidney disease caused by collagen IV mutations that interfere with the formation and deposition of the α3α4α5 protomer into the glomerular basement membrane. In this issue, Yu et al. show that the chemical chaperone tauroursodeoxycholic acid prevented kidney structural changes and function decline in mice with a pathogenic missense Col4a3 mutation by increasing mutant α3α4α5 protomer glomerular basement membrane deposition and preventing podocyte apoptosis induced by endoplasmic reticulum stress.

Asunto(s)

Autoantígenos , Colágeno Tipo IV , Membrana Basal Glomerular , Nefritis Hereditaria , Ácido Tauroquenodesoxicólico , Nefritis Hereditaria/genética , Nefritis Hereditaria/tratamiento farmacológico , Nefritis Hereditaria/patología , Nefritis Hereditaria/metabolismo , Animales , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Ácido Tauroquenodesoxicólico/farmacología , Ácido Tauroquenodesoxicólico/uso terapéutico , Ratones , Membrana Basal Glomerular/patología , Membrana Basal Glomerular/efectos de los fármacos , Humanos , Autoantígenos/genética , Autoantígenos/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Podocitos/efectos de los fármacos , Podocitos/patología , Podocitos/metabolismo , Mutación Missense , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo

RESUMEN

Human telomerase assembly is a highly dynamic process. Using biochemical approaches, we find that LARP3 and LARP7/MePCE are involved in the early stage of human telomerase RNA (hTR) and that their binding to RNA is destabilized when the mature form is produced. LARP3 plays a negative role in preventing the processing of the 3'-extended long (exL) form and the binding of LARP7 and MePCE. Interestingly, the tertiary structure of the exL form prevents LARP3 binding and facilitates hTR biogenesis. Furthermore, low levels of LARP3 promote hTR maturation, increase telomerase activity, and elongate telomeres. LARP7 and MePCE depletion inhibits the conversion of the 3'-extended short (exS) form into mature hTR and the cytoplasmic accumulation of hTR, resulting in telomere shortening. Taken together our data suggest that LARP3 and LARP7/MePCE mediate the processing of hTR precursors and regulate the production of functional telomerase.

Asunto(s)

Autoantígenos , ARN , Ribonucleoproteínas , Antígeno SS-B , Telomerasa , Humanos , Autoantígenos/metabolismo , Autoantígenos/genética , Células HeLa , Unión Proteica , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , ARN/metabolismo , ARN/genética , Telomerasa/metabolismo , Telomerasa/genética , Telómero/metabolismo , Telómero/genética , Acortamiento del Telómero

RESUMEN

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Asunto(s)

Membrana Basal , Colágeno Tipo IV , Modelos Animales de Enfermedad , Ratones Noqueados , Nefritis Hereditaria , Animales , Nefritis Hereditaria/patología , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/deficiencia , Ratones , Membrana Basal/metabolismo , Membrana Basal/patología , Membrana Basal/ultraestructura , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Microscopía Electrónica de Transmisión , Ratones Endogámicos C57BL , Cápsula del Cristalino/metabolismo , Cápsula del Cristalino/patología , Cápsula del Cristalino/ultraestructura , Epitelio Corneal/patología , Epitelio Corneal/ultraestructura , Epitelio Corneal/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Retina/patología , Retina/metabolismo , Retina/ultraestructura , Autoantígenos/genética , Autoantígenos/metabolismo , Células Ependimogliales/patología , Células Ependimogliales/metabolismo , Células Ependimogliales/ultraestructura , Inmunohistoquímica , Masculino

RESUMEN

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Asunto(s)

Artritis Reumatoide , Autoantígenos , Chaperonina 60 , Centro Germinal , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Animales , Humanos , Ratones , Autoantígenos/inmunología , Autoantígenos/genética , Centro Germinal/inmunología , Centro Germinal/patología , Chaperonina 60/inmunología , Chaperonina 60/genética , Autoanticuerpos/inmunología , Autoinmunidad , Masculino , Sinoviocitos/inmunología , Sinoviocitos/patología , Sinoviocitos/metabolismo , Artritis Experimental/inmunología , Artritis Experimental/patología , Femenino , Linfocitos B/inmunología , Linfocitos B/patología , Estructuras Linfoides Terciarias/inmunología , Estructuras Linfoides Terciarias/patología

RESUMEN

Bullous pemphigoid (BP) is a type 2 inflammation- and immunity-driven skin disease, yet a comprehensive understanding of the immune landscape, particularly immune-stromal crosstalk in BP, remains elusive. Herein, using single-cell RNA sequencing (scRNA-seq) and in vitro functional analyzes, we pinpoint Th2 cells, dendritic cells (DCs), and fibroblasts as crucial cell populations. The IL13-IL13RA1 ligand-receptor pair is identified as the most significant mediator of immune-stromal crosstalk in BP. Notably, fibroblasts and DCs expressing IL13RA1 respond to IL13-secreting Th2 cells, thereby amplifying Th2 cell-mediated cascade responses, which occurs through the specific upregulation of PLA2G2A in fibroblasts and CCL17 in myeloid cells, creating a positive feedback loop integral to immune-stromal crosstalk. Furthermore, PLA2G2A and CCL17 contribute to an increased titer of pathogenic anti-BP180-NC16A autoantibodies in BP patients. Our work provides a comprehensive insight into BP pathogenesis and shows a mechanism governing immune-stromal interactions, providing potential avenues for future therapeutic research.

Asunto(s)

Quimiocina CCL17 , Células Dendríticas , Fibroblastos , Penfigoide Ampolloso , Análisis de la Célula Individual , Células Th2 , Humanos , Penfigoide Ampolloso/inmunología , Penfigoide Ampolloso/genética , Análisis de la Célula Individual/métodos , Fibroblastos/metabolismo , Fibroblastos/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Células Th2/inmunología , Autoanticuerpos/inmunología , Transcriptoma , Interleucina-13/metabolismo , Interleucina-13/genética , Interleucina-13/inmunología , Colágenos no Fibrilares/inmunología , Colágenos no Fibrilares/genética , Colágenos no Fibrilares/metabolismo , Inflamación/inmunología , Inflamación/genética , Inflamación/metabolismo , Perfilación de la Expresión Génica/métodos , Masculino , Femenino , Autoantígenos/inmunología , Autoantígenos/metabolismo , Autoantígenos/genética , Colágeno Tipo XVII , Células Mieloides/metabolismo , Células Mieloides/inmunología , Células del Estroma/metabolismo , Células del Estroma/inmunología

RESUMEN

Background and Objectives: Congenital thyroid dyshormonogenesis is caused by alterations in the synthesis of thyroid hormones in a newborn. Additionally, 10 to 20% of these cases are hereditary, caused by defects in proteins involved in hormonal synthesis. One of the most common causes is mutations in the thyroid peroxidase (TPO) enzyme gene, an autosomal recessive disease. We aimed to detect mutations of the TPO gene in 12 Chilean patients with congenital hypothyroidism due to dyshormonogenesis (CHD) and to characterize these patients clinically and molecularly. Materials and Methods: Twelve patients under 20 years of age with CHD, controlled at San Juan de Dios Hospital in Santiago, Chile, were selected according to the inclusion criteria: elevated neonatal TSH, persistent hypothyroidism, and thyroid normotopic by imaging study. Those with deafness, Down syndrome, and central or transient congenital hypothyroidism were excluded. Blood samples were taken for DNA extraction, and the 17 exons and exon-intron junctions of the TPO gene were amplified by PCR. The PCR products were sequenced by Sanger. Results: Two possibly pathogenic mutations of the TPO gene were detected: c.2242G>A (p.Val748Met) and c.1103C>T (p.Pro368Leu). These mutations were detected in 2 of 12 patients (16.6%): 1 was compound heterozygous c.1103C>T/c.2242G>A, and the other was heterozygous for c.2242G>A. In the diagnostic confirmation test, both patients presented diffuse hyper-uptake goiter on thyroid scintigraphy and high TSH in venous blood (>190 uIU/mL). Conclusions: The frequency of patients with possibly pathogenic mutations in TPO with CHD was 16.6%. Its study would allow for genetic counseling to be offered to the families of affected patients.

Asunto(s)

Hipotiroidismo Congénito , Yoduro Peroxidasa , Proteínas de Unión a Hierro , Mutación , Humanos , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/sangre , Chile , Yoduro Peroxidasa/genética , Femenino , Masculino , Proteínas de Unión a Hierro/genética , Autoantígenos/genética , Lactante , Niño , Adolescente , Preescolar , Recién Nacido , Disgenesias Tiroideas/genética , Disgenesias Tiroideas/complicaciones , Disgenesias Tiroideas/sangre

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

BACKGROUND: Alport syndrome (AS) is an inherited nephropathy caused by mutations in the type IV collagen genes. It is clinically characterized by damage to the eyes, ears and kidneys. Diagnosis of AS is hampered by its atypical clinical picture, particularly when the typical features, include persistent hematuria and microscopic changes in the glomerular basement membrane (GBM), are the only clinical manifestations in the patient. METHODS: We screened 10 families with suspected AS using whole exome sequencing (WES) and analyzed the harmfulness, conservation, and protein structure changes of mutated genes. In further, we performed in vitro functional analysis of two missense mutations in the COL4A5 gene (c.2359G > C, p.G787R and c.2605G > A, p.G869R). RESULTS: We identified 11 pathogenic variants in the type IV collagen genes (COL4A3, COL4A4 and COL4A5). These pathogenic variants include eight missense mutations, two nonsense mutations and one frameshift mutation. Notably, Family 2 had digenic mutations in the COL4A3 (p.G1170A) and UMOD genes (p.M229K). Family 3 had a digenic missense mutation (p.G997E) in COL4A3 and a frameshift mutation (p.P502L fs*151) in COL4A4. To our knowledge, four of the 11 mutations are novel mutations. In addition, we found that COL4A5 mutation relation mRNA levels were significantly decreased in HEK 293 T cell compared to control, while the cellular localization remained the same. CONCLUSIONS: Our research expands the spectrum of COL4A3-5 pathogenic variants, which is helpful for clinical and scientific research.

Asunto(s)

Autoantígenos , Colágeno Tipo IV , Nefritis Hereditaria , Linaje , Humanos , Nefritis Hereditaria/genética , Nefritis Hereditaria/patología , Colágeno Tipo IV/genética , Autoantígenos/genética , Femenino , Masculino , Adulto , Mutación , Secuenciación del Exoma , Persona de Mediana Edad , Mutación Missense , Células HEK293

RESUMEN

Cellular communication (CC) influences tumor development by mediating intercellular junctions between cells. However, the role and underlying mechanisms of CC in malignant transformation remain unknown. Here, we investigated the spatiotemporal heterogeneity of CC molecular expression during malignant transformation. It was found that although both tight junctions (TJs) and gap junctions (GJs) were involved in maintaining the tumor microenvironment (TME), they exhibited opposite characteristics. Mechanistically, for epithelial cells (parenchymal component), the expression of TJ molecules consistently decreased during normal-cancer transformation and is a potential oncogenic factor. For fibroblasts (mesenchymal component), the expression of GJs consistently increased during normal-cancer transformation and is a potential oncogenic factor. In addition, the molecular profiles of TJs and GJs were used to stratify colorectal cancer (CRC) patients, where subtypes characterized by high GJ levels and low TJ levels exhibited enhanced mesenchymal signals. Importantly, we propose that leiomodin 1 (LMOD1) is biphasic, with features of both TJs and GJs. LMOD1 not only promotes the activation of cancer-associated fibroblasts (CAFs) but also inhibits the Epithelial-mesenchymal transition (EMT) program in cancer cells. In conclusion, these findings demonstrate the molecular heterogeneity of CC and provide new insights into further understanding of TME heterogeneity.

Asunto(s)

Fibroblastos Asociados al Cáncer , Comunicación Celular , Neoplasias Colorrectales , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Microambiente Tumoral , Animales , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Transición Epitelial-Mesenquimal/genética , Uniones Comunicantes/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Análisis Espacio-Temporal , Uniones Estrechas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo

RESUMEN

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and it lacks specific therapeutic targets and effective treatment protocols. By analyzing a proteomic TNBC dataset, we found significant upregulation of sideroflexin 1 (SFXN1) in tumor tissues. However, the precise function of SFXN1 in TNBC remains unclear. Immunoblotting was performed to determine SFXN1 expression levels. Label-free quantitative proteomics and liquid chromatography-tandem mass spectrometry were used to identify the downstream targets of SFXN1. Mechanistic studies of SFXN1 and cellular inhibitor of PP2A (CIP2A) were performed using immunoblotting, immunofluorescence staining, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Functional experiments were used to investigate the role of SFXN1 in TNBC cells. SFXN1 was significantly overexpressed in TNBC tumor tissues and was associated with unfavorable outcomes in patients with TNBC. Functional experiments demonstrated that SFXN1 promoted TNBC growth and metastasis in vitro and in vivo. Mechanistic studies revealed that SFXN1 promoted TNBC progression by inhibiting the autophagy receptor TOLLIP (toll interacting protein)-mediated autophagic degradation of CIP2A. The pro-tumorigenic effect of SFXN1 overexpression was partially prevented by lapatinib-mediated inhibition of the CIP2A/PP2A/p-AKT pathway. These findings may provide a new targeted therapy for patients with TNBC.

Asunto(s)

Autoantígenos , Autofagia , Proteínas de Transporte de Catión , Lapatinib , Proteínas de la Membrana , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Antineoplásicos/farmacología , Autoantígenos/metabolismo , Autoantígenos/genética , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Lapatinib/farmacología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo

RESUMEN

TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis. Here we show that ASRGL1 expression was diminished in ALS brain samples by RNA sequencing, immunohistochemistry, and western blotting. TDP-43 and ASRGL1 colocalized in neurons but, in the absence of ASRGL1, TDP-43 aggregated in the cytoplasm. TDP-43 was found to be prone to isoaspartate formation and a substrate for ASRGL1. ASRGL1 silencing triggered accumulation of misfolded, fragmented, phosphorylated and mislocalized TDP-43 in cultured neurons and motor cortex of female mice. Overexpression of ASRGL1 restored neuronal viability. Overexpression of HML-2 led to ASRGL1 silencing. Loss of ASRGL1 leading to TDP-43 aggregation may be a critical mechanism in ALS pathophysiology.

Asunto(s)

Esclerosis Amiotrófica Lateral , Asparaginasa , Proteínas de Unión al ADN , Neuronas , Proteinopatías TDP-43 , Animales , Femenino , Humanos , Masculino , Ratones , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Asparaginasa/genética , Asparaginasa/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Corteza Motora/metabolismo , Corteza Motora/patología , Neuronas/metabolismo , Neuronas/patología , Proteinopatías TDP-43/metabolismo , Proteinopatías TDP-43/patología , Proteinopatías TDP-43/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo

RESUMEN

BACKGROUND: Early embryonic arrest and fragmentation (EEAF) is a common cause of female infertility, but the genetic causes remain to be largely unknown. CIP2A encodes the cellular inhibitor of PP2A, playing a crucial role in mitosis and mouse oocyte meiosis. METHODS: Exome sequencing and Sanger sequencing were performed to identify candidate causative genes in patients with EEAF. The pathogenicity of the CIP2A variant was assessed and confirmed in cultured cell lines and human oocytes through Western blotting, semi-quantitative RT-PCR, TUNEL staining, and fluorescence localization analysis. FINDINGS: We identified CIP2A (c.1510C > T, p.L504F) as a novel disease-causing gene in human EEAF from a consanguineous family. L504 is highly conserved throughout evolution. The CIP2A variant (c.1510C > T, p.L504F) reduced the expression level of the mutant CIP2A protein, leading to the abnormal aggregation of mutant CIP2A protein and cell apoptosis. Abnormal aggregation of CIP2A protein and chromosomal dispersion occurred in the patient's oocytes and early embryos. We further replicated the patient phenotype by knockdown CIP2A in human oocytes. Additionally, CIP2A deficiency resulted in decreased levels of phosphorylated ERK1/2. INTERPRETATION: We first found that the CIP2A loss-of-function variant associate with female infertility characterized by EEAF. Our findings suggest the uniqueness and importance of CIP2A gene in human oocyte and early embryo development. FUNDING: This work was supported by National Key Research and Development Program of China (2023YFC2706302), the National Natural Science Foundation of China (81000079, 81170165, and 81870959), the HUST Academic Frontier Youth Team (2016QYTD02), and the Key Research of Huazhong University of Science and Technology, Tongji Hospital (2022A20).

Asunto(s)

Autoantígenos , Infertilidad Femenina , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Oocitos , Humanos , Femenino , Autoantígenos/genética , Autoantígenos/metabolismo , Infertilidad Femenina/genética , Infertilidad Femenina/patología , Infertilidad Femenina/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Apoptosis/genética , Mutación con Pérdida de Función , Adulto , Secuenciación del Exoma , Animales , Linaje , Ratones

RESUMEN

Alport syndrome and autosomal dominant polycystic kidney disease are monogenic causes of chronic kidney disease and end-stage kidney failure. We present a case of a man in his 60s with progressive chronic kidney disease, bilateral sensorineural hearing loss and multiple renal cysts. Genetic analysis revealed a heterozygous variant in COL4A3 (linked to Alport syndrome) and in the GANAB gene (associated with a milder form of autosomal dominant polycystic kidney disease). Although each variant confers a mild risk of developing end-stage kidney disease, the patient presented a pronounced and accelerated progression of chronic kidney disease, which goes beyond what would be predicted by adding up their individual effects. This suggests a potential synergic effect of both variants, which warrants further investigation.

Asunto(s)

Colágeno Tipo IV , Nefritis Hereditaria , Riñón Poliquístico Autosómico Dominante , Humanos , Nefritis Hereditaria/genética , Nefritis Hereditaria/complicaciones , Nefritis Hereditaria/diagnóstico , Masculino , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/complicaciones , Colágeno Tipo IV/genética , Persona de Mediana Edad , Autoantígenos/genética , Progresión de la Enfermedad , Fallo Renal Crónico/genética , Fallo Renal Crónico/etiología , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/diagnóstico

RESUMEN

COL4A3/A4/A5 mutations have been identified as critical causes of Alport syndrome and other genetic chronic kidney diseases. However, the underlying pathogenesis remains unclear, and specific treatments are lacking. Here, we constructed a transgenic Alport syndrome mouse model by generating a mutation (Col4a3 p.G799R) identified previously from one large Alport syndrome family into mice. We observed that the mutation caused a pathological decrease in intracellular and secreted collagen IV α3α4α5 heterotrimers. The mutant collagen IV α3 chains abnormally accumulated in the endoplasmic reticulum and exhibited defective secretion, leading to persistent endoplasmic reticulum stress in vivo and in vitro. RNA-seq analysis revealed that the MyD88/p38 MAPK pathway plays key roles in mediating subsequent inflammation and apoptosis signaling activation. Treatment with tauroursodeoxycholic acid, a chemical chaperone drug that functions as an endoplasmic reticulum stress inhibitor, effectively suppressed endoplasmic reticulum stress, promoted secretion of the α3 chains, and inhibited the activation of the MyD88/p38 MAPK pathway. Tauroursodeoxycholic acid treatment significantly improved kidney function in vivo. These results partly clarified the pathogenesis of kidney injuries associated with Alport syndrome, especially in glomeruli, and suggested that tauroursodeoxycholic acid might be useful for the early clinical treatment of Alport syndrome.

Asunto(s)

Colágeno Tipo IV , Estrés del Retículo Endoplásmico , Mutación , Nefritis Hereditaria , Ácido Tauroquenodesoxicólico , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Humanos , Masculino , Ratones , Apoptosis/efectos de los fármacos , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Riñón/patología , Riñón/efectos de los fármacos , Riñón/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Nefritis Hereditaria/genética , Nefritis Hereditaria/tratamiento farmacológico , Nefritis Hereditaria/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido Tauroquenodesoxicólico/farmacología , Ácido Tauroquenodesoxicólico/uso terapéutico

RESUMEN

Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

Asunto(s)

Cilios , Ribonucleoproteínas , Tubulina (Proteína) , Proteínas de Xenopus , Xenopus laevis , Cilios/metabolismo , Animales , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Humanos , Antígeno SS-B , Autoantígenos/metabolismo , Autoantígenos/genética , Unión Proteica , Axonema/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética

RESUMEN

One of the probable hypotheses for the onset of autoimmunity is molecular mimicry. This study aimed to determine the HLA-II risk alleles for developing Hashimoto's thyroiditis (HT) in order to analyze the molecular homology between candidate pathogen-derived epitopes and potentially self-antigens (thyroid peroxidase, TPO) based on the presence of HLA risk alleles. HLA-DRB1/-DQB1 genotyping was performed in 100 HT patients and 330 ethnically matched healthy controls to determine the predisposing/protective alleles for HT disease. Then, in silico analysis was conducted to examine the sequence homology between epitopes derived from autoantigens and four potentially relevant pathogens and their binding capacities to HLA risk alleles based on peptide docking analysis. We identified HLA-DRB1*03:01, *04:02, *04:05, and *11:04 as predisposing alleles and DRB1*13:01 as a potentially predictive allele for HT disease. Also, DRB1*11:04 ~ DQB1*03:01 (Pc = 0.002; OR, 3.97) and DRB1*03:01 ~ DQB1*02:01 (Pc = 0.004; OR, 2.24) haplotypes conferred a predisposing role for HT. Based on logistic regression analysis, carrying risk alleles increased the risk of HT development 4.5 times in our population (P = 7.09E-10). Also, ROC curve analysis revealed a high predictive power of those risk alleles for discrimination of the susceptible from healthy individuals (AUC, 0.70; P = 6.6E-10). Analysis of peptide sequence homology between epitopes of TPO and epitopes derived from four candidate microorganisms revealed a homology between envelop glycoprotein D of herpes virus and sequence 151-199 of TPO with remarkable binding capacity to HLA-DRB1*03:01 allele. Our findings indicate the increased risk of developing HT in those individuals carrying HLA risk alleles which can also be related to herpes virus infection.

Asunto(s)

Alelos , Autoantígenos , Epítopos , Predisposición Genética a la Enfermedad , Cadenas beta de HLA-DQ , Cadenas HLA-DRB1 , Enfermedad de Hashimoto , Humanos , Masculino , Femenino , Enfermedad de Hashimoto/genética , Enfermedad de Hashimoto/inmunología , Adulto , Irán , Cadenas HLA-DRB1/genética , Cadenas beta de HLA-DQ/genética , Autoantígenos/inmunología , Autoantígenos/genética , Epítopos/inmunología , Epítopos/genética , Persona de Mediana Edad , Estudios de Casos y Controles , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/inmunología , Haplotipos , Genotipo , Frecuencia de los Genes

RESUMEN

Recently, the progression of gastric cancer (GC), as one of the most ordinary malignant tumors, has been reported to be associated with circular RNAs. This study aimed to identify the role of circular RNA_LARP4 in GC. We performed real-time quantitative polymerase chain reaction (RT-qPCR) in 46 paired GC patients and GC cell lines to detect the expression of circular RNA_LARP4. Moreover, the role of circular RNA_LARP4 in GC proliferation was identified through proliferation assay and colony formation assay, while the role of circular RNA_LARP4 in GC metastasis was measured through scratch wound assay and transwell assay. Furthermore, the potential targets of circular RNA_LARP4 were predicted through bioinformatics methods and further identified by western blot assay and RT-qPCR. Circular RNA_LARP4 expression was remarkably lower in GC tissues compared with that in adjacent samples. Besides, cell proliferation of GC was inhibited after overexpression of circular RNA_LARP4, while cell migration and invasion of GC was inhibited after overexpression of circular RNA_LARP4. Furthermore, Upstream frameshift 1 (UPF1) was predicted as the potential target of circular RNA_LARP4 and was upregulated via overexpression of circular RNA_LARP4 in GC. Circular RNA_LARP4 inhibits GC cell proliferation and metastasis via targeting UPF1 in vitro, which might provide a new tumor suppressor in GC development.

Asunto(s)

Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , ARN Circular , Neoplasias Gástricas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Invasividad Neoplásica/genética , Metástasis de la Neoplasia , ARN/genética , ARN/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Antígeno SS-B , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Regulación hacia Arriba/genética