Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types.<br/

VCP/p97-associated proteins are binders and debranching enzymes of K48–K63-branched ubiquitin chains

Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48–K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48–K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48–K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48–K63-linked Ub, these results suggest a function for K48–K63-branched chains in VCP/p97-related processes

VCP/p97-associated proteins are binders and debranching enzymes of K48–K63-branched ubiquitin chains

Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48–K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48–K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48–K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48–K63-linked Ub, these results suggest a function for K48–K63-branched chains in VCP/p97-related processes

Single-cell analysis reveals specific neuronal transition during mouse corticogenesis

Background: Currently, the mechanism(s) underlying corticogenesis is still under characterization.Methods: We curated the most comprehensive single-cell RNA-seq (scRNA-seq) datasets from mouse and human fetal cortexes for data analysis and confirmed the findings with co-immunostaining experiments.Results: By analyzing the developmental trajectories with scRNA-seq datasets in mice, we identified a specific developmental sub-path contributed by a cell-population expressing both deep- and upper-layer neurons (DLNs and ULNs) specific markers, which occurred on E13.5 but was absent in adults. In this cell-population, the percentages of cells expressing DLN and ULN markers decreased and increased, respectively, during the development suggesting direct neuronal transition (namely D-T-U). Whilst genes significantly highly/uniquely expressed in D-T-U cell population were significantly enriched in PTN/MDK signaling pathways related to cell migration. Both findings were further confirmed by co-immunostaining with DLNs, ULNs and D-T-U specific markers across different timepoints. Furthermore, six genes (co-expressed with D-T-U specific markers in mice) showing a potential opposite temporal expression between human and mouse during fetal cortical development were associated with neuronal migration and cognitive functions. In adult prefrontal cortexes (PFC), D-T-U specific genes were expressed in neurons from different layers between humans and mice.Conclusion: Our study characterizes a specific cell population D-T-U showing direct DLNs to ULNs neuronal transition and migration during fetal cortical development in mice. It is potentially associated with the difference of cortical development in humans and mice

Listeria pathogenesis and molecular virulence determinants

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research

Metal-organic frameworks induce autophagy in mouse embryonic fibroblast cells

Autophagy is the lysosomal-dependent degradation process of intracellular substances in adaptation to environmental or developmental changes. It plays an essential role in maintaining cellular homeostasis while its dysfunction is involved in various human diseases. The regulation of autophagy has attracted more and more attention with the promise for improving treatment of diseases as a potential therapeutic target. Metal-organic frameworks (MOFs), as emerging biomaterials, have been investigated in the biological and biomedical fields in recent years. Therefore, it is interesting and significant to study the effects of MOFs on living cells from safety aspects as well as the therapeutic viewpoint, especially their effects on autophagy which have not been reported yet. In this study, the effects of Fe-MIL-101_NH2 on mouse embryonic fibroblasts (MEFs) were investigated and the potential applications of these nanoparticles in the regulation of autophagy were explored. Our results demonstrated that Fe-MIL-101_NH2 induced cytoprotective autophagy in MEFs instead of cytotoxicity. The activation of autophagy kept reactive oxygen species from accumulating, which protected MEFs from apoptosis. Further exploration of the possible mechanisms of MOF-induced autophagy revealed that the inhibition of mTOR pathway as well as the enhancement of Becline1 and Atg5 contributed to autophagy induction. Our study uncovered the autophagic effects and mechanistic insights of MOFs, which will be beneficial and meaningful to the safety evaluation and the reasonable and effective usage of MOFs

Agonist-induced aggregation of Chinese hamster ovary cells coexpressing the human receptors for fibrinogen (integrin alphaIIbbeta3) and the platelet-activating factor: dissociation between adhesion and aggregation

9 páginas, 10 figuras, 1 tabla -- PAGS nros. 2819-2827This work reports the establishment of a Chinese hamster ovary (CHO) cell line stably coexpressing the human αIIbβ3 integrin and the platelet-activating factor receptor (PAFR). These cells aggregate in response to PAF in a Ca++, αIIbβ3, and soluble fibrinogen (Fg)–dependent manner that is prevented by PAF antagonists or αIIbβ3 blockade. The aggregating response is accompanied by enhanced binding of fibrinogen and the activation-dependent IgM PAC1. This model has permitted us to identify, for the first time, intracellular signals distinctly associated with either αIIbβ3-mediated adhesion or aggregation. Nonreceptor activation of protein kinase C (PKC) by phorbol ester produced cellular adhesion and spreading onto immobilized Fg, but it was not a sufficient signal to provoke cellular aggregation. Moreover, inhibition of PKC impeded the PAF stimulation of cellular adhesion, whereas the aggregation was not prevented. The PAF-induced cellular aggregation was distinctly associated with signaling events arising from the liganded Fg receptor and the agonist-induced stimulation of a calcium/calmodulin-dependent signaling pathway. Sustained tyrosine phosphorylation of both mitogen-activated protein kinase (MAPK) and an approximately 100-kd protein was associated with the PAF-induced aggregation, whereas phosphorylation of focal adhesion kinase (FAK) was preferably associated with cellular adherence and spreading onto immobilized Fg.Supported in part by grants from the Dirección General de Investigación Cientı́fica y Técnica (DGICYT PB97-1240, SAF 2000-0127 and DGICYT PM97-0016), Fondo de Investigaciones Sanitarias (96/2014), and Comunidad Autónoma de Madrid (08.4/0031/1998). L.S. was supported by a grant-in-aid from the Agencia Española de Cooperación Internacional (AECI)Peer reviewe

Facile one-step solvothermal synthesis of a luminescent europium metal-organic framework for rapid and selective sensing of uranyl ions

A simple, cost-effective, and portable uranium sensory material with adequate selectivity is increasingly urgent and would be of great importance in environmental monitoring of radionuclides. Herein, we report a novel luminescent europium metal-organic framework (Eu-MOF) with plenty of Lewis basic sites for binding uranyl ions (UO2 (2+)), the most common form of uranium in solution, through a facile one-step solvothermal synthetic route. The mesoporous structure consists of europium nodes and flexible nitrogen-containing ligands with a 29.2 x 20.5 (2) channel along the c-axis. Furthermore, the obtained material displays characteristic fluorescence of trivalent Eu3+ and could be applied as a turn-off sensory probe targeting UO2 (2+) in solution. Differential fluorescent quenching occurred upon a series of potential interfering ions compared to UO2 (2+) and the detection limit as low as 0.9 mu M was achieved with a rapid response

Ribosomal proteins are targets for the NEDD8 pathway

Identification of the molecular targets for post-translational modifications is an important step for explaining the regulated pathways. The ubiquitin-like molecule NEDD8 is implicated in the regulation of cell proliferation, viability and development. By combining proteomics and in vivo NEDDylation assays, we identified a subset of ribosomal proteins as novel targets for the NEDD8 pathway. We further show that the lack of NEDDylation in cells causes ribosomal protein instability. Our studies identify a novel and specific role of the NEDD8 pathway in protecting a subset of ribosomal proteins from destabilization