RESUMEN
Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60-80% of cases1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria2. Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes3. Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide. These helical assemblies organize into densely packed protein rungs with minimal inter-rung connectivity, and exhibit nucleotide-dependent dimerization of the GTPase domains-a hallmark of the dynamin superfamily of proteins4. OPA1 also contains several unique secondary structures in the paddle domain that strengthen its membrane association, including membrane-inserting helices. The structural features identified in this study shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly and membrane interactions. Furthermore, mutations that disrupt the assembly interfaces and membrane binding of OPA1 cause mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions.
Asunto(s)
Microscopía por Crioelectrón , GTP Fosfohidrolasas , Mitocondrias , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/ultraestructura , Fusión de Membrana , Mitocondrias/enzimología , Mitocondrias/metabolismo , Mitocondrias/patología , Dinámicas Mitocondriales , Membranas Mitocondriales/metabolismo , Mutación , Nucleótidos/metabolismo , Unión Proteica/genética , Dominios Proteicos , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , HumanosRESUMEN
Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.
Asunto(s)
Cisteína , Mucinas , Mucinas/metabolismo , Cisteína/metabolismo , Transporte Biológico , Vesículas Secretoras/metabolismoRESUMEN
Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal , Leucemia Mieloide Aguda , Animales , Humanos , Macaca mulatta , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patología , Trombopoyesis , FenotipoRESUMEN
Mucins are large, highly glycosylated transmembrane and secreted proteins that line and protect epithelial surfaces. However, the details of mucin biosynthesis and packaging in vivo are largely unknown. Here, we demonstrate that multiple distinct mucins undergo intragranular restructuring during secretory granule maturation in vivo, forming unique structures that are spatially segregated within the same granule. We further identify temporally-regulated genes that influence mucin restructuring, including those controlling pH (Vha16-1), Ca2+ ions (fwe) and Cl- ions (Clic and ClC-c). Finally, we show that altered mucin glycosylation influences the dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular segregation of mucins through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct mucins are efficiently packaged into and secreted from secretory granules may provide insight into diseases resulting from defects in mucin secretion.
Asunto(s)
Mucinas , Vesículas Secretoras , Gránulos Citoplasmáticos/metabolismo , Glicosilación , Mucinas/metabolismo , Vesículas Secretoras/metabolismoRESUMEN
The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.
Asunto(s)
SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Fusión Celular , Línea Celular , Furina/metabolismo , Células Gigantes , Glicosilación , Humanos , N-Acetilgalactosaminiltransferasas/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
The COVID-19 global pandemic has underscored the need to understand how viruses and other pathogens are able to infect and replicate within the respiratory system. Recent studies have highlighted the role of highly O-glycosylated mucins in the protection of the respiratory system as well as how mucin-type O-glycosylation may be able to modify viral infectivity. Therefore, we set out to identify the specific genes controlling mucin-type O-glycosylation throughout the mouse respiratory system as well as determine how their expression and the expression of respiratory mucins is influenced by infection or injury. Here, we show that certain mucins and members of the Galnt family are abundantly expressed in specific respiratory tissues/cells and demonstrate unique patterns of O-glycosylation across diverse respiratory tissues. Moreover, we find that the expression of certain Galnts and mucins is altered during lung infection and injury in experimental mice challenged with infectious agents, toxins, and allergens. Finally, we examine gene expression changes of Galnts and mucins in a mouse model of SARS-CoV-2 infection. Our work provides foundational knowledge regarding the specific expression of Galnt enzyme family members and mucins throughout the respiratory system, and how their expression is altered upon lung infection and injury.
Asunto(s)
COVID-19 , Mucinas , Animales , Ratones , Mucinas/genética , Mucinas/metabolismo , Glicosilación , COVID-19/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Sistema Respiratorio/metabolismoRESUMEN
Mucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation, and organ growth. O-Glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of GalNAc onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain two functional domains: a catalytic domain and a C-terminal ricin-like lectin domain comprised of three potential GalNAc recognition repeats termed α, ß, and γ. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing transfer of GalNAc, whereas the lectin domain recognizes more distant extant GalNAc on previously glycosylated substrates. We previously demonstrated a novel role for the α repeat of lectin domain in influencing charged peptide preferences. Here, we further interrogate how the differentially spliced α repeat of the PGANT9A and PGANT9B O-glycosyltransferases confers distinct preferences for a variety of endogenous substrates. Through biochemical analyses and in silico modeling using preferred substrates, we find that a combination of charged residues within the α repeat and charged residues in the flexible gating loop of the catalytic domain distinctively influence the peptide substrate preferences of each splice variant. Moreover, PGANT9A and PGANT9B also display unique glycopeptide preferences. These data illustrate how changes within the noncatalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates. Overall, our results elucidate a novel mechanism for modulating substrate preferences of O-glycosyltransferases via alternative splicing within specific subregions of functional domains.
Asunto(s)
Simulación por Computador , Proteínas de Drosophila/química , Glicopéptidos/química , Glicosiltransferasas/química , Empalme Alternativo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Glicopéptidos/genética , Glicosilación , Glicosiltransferasas/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Especificidad por SustratoRESUMEN
An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de la Matriz Extracelular/metabolismo , Tracto Gastrointestinal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Tracto Gastrointestinal/anatomía & histología , Tracto Gastrointestinal/embriología , Glicoproteínas/genética , MorfogénesisRESUMEN
The secreted mucus layer that lines and protects epithelial cells is conserved across diverse species. While the exact composition of this protective layer varies between organisms, certain elements are conserved, including proteins that are heavily decorated with N-acetylgalactosamine-based sugars linked to serines or threonines (O-linked glycosylation). These heavily O-glycosylated proteins, known as mucins, exist in many forms and are able to form hydrated gel-like structures that coat epithelial surfaces. In vivo studies in diverse organisms have highlighted the importance of both the mucin proteins as well as their constituent O-glycans in the protection and health of internal epithelia. Here, we summarize in vivo approaches that have shed light on the synthesis and function of these essential components of mucus.
Asunto(s)
Mucinas , Moco , Células Epiteliales/metabolismo , Glicosilación , Humanos , Mucinas/química , Mucinas/metabolismo , Moco/metabolismo , PolisacáridosRESUMEN
In epithelia, specialized tricellular junctions (TCJs) mediate cell contacts at three-cell vertices. TCJs are fundamental to epithelial biology and disease, but only a few TCJ components are known, and how they assemble at tricellular vertices is not understood. Here we describe a transmembrane protein, Anakonda (Aka), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss of Aka causes epithelial barrier defects associated with irregular TCJ structure and geometry, suggesting that Aka organizes cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiates TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain has an unusual tripartite repeat structure that may mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/citología , Epitelio/crecimiento & desarrollo , Uniones Intercelulares/fisiología , Uniones Estrechas/fisiología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/metabolismo , Epitelio/metabolismo , Immunoblotting , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas , Secuencias Repetitivas de AminoácidoRESUMEN
Vital vertebrate organs are protected from the external environment by a barrier that to a large extent consists of mucins. These proteins are characterized by poorly conserved repeated sequences that are rich in prolines and potentially glycosylated threonines and serines (PTS). We have now used the characteristics of the PTS repeat domain to identify Drosophila mucins in a simple bioinformatics approach. Searching the predicted protein database for proteins with at least 4 repeats and a high ST content, more than 30 mucin-like proteins were identified, ranging from 300-23000 amino acids in length. We find that Drosophila mucins are present at all stages of the fly life cycle, and that their transcripts localize to selective organs analogous to sites of vertebrate mucin expression. The results could allow for addressing basic questions about human mucin-related diseases in this model system. Additionally, many of the mucins are expressed in selective tissues during embryogenesis, thus revealing new potential functions for mucins as apical matrix components during organ morphogenesis.