Structural and functional characterization of human ERp44: a closer look at a member of PDI family regulating protein quality control in the early secretory pathway

The Endoplasmic Reticulum (ER) is the site of folding and assembly of secretory proteins. Fidelity of protein-based intracellular communication is guaranteed by protein quality control mechanisms located at the Early Secretory Compartment (ESC), which restricts forward transport to native proteins. ERp44 plays a key role in the Thiol-Mediated Retention (TMR) of variety of client proteins (Ero1, SUMF1, Adiponectin, IgM) thus regulating their transport and localization. Little is known about the molecular mechanisms of ERp44-TMR and how it is regulated in living cells. Hence, the overall aim of this work is to investigate the structure-function relationship of ERp44. In collaboration with Wang's group the crystal structure of ERp44 was determined. The structure of ERp44 most likely represents a non-reactive conformation of the protein. Indeed, Cys29 is shielded from the bulk solvent by C-terminal tail and almost inaccessible for the formation of intermolecular disulfide bonds with client proteins. Based on the obtained structural data and functional studies, a panel of mutants of ERp44 has been characterized in order to understand how C-terminal tail rearrangements expose substrate binding site, thus modulating substrates binding/ release in view of its role in TMR. Moreover, the pH gradient between ESC organelles was investigated as major determinant of C-terminal tail rearrangements. Given the known pH differences in the ESC, the data support the hypothesis of a role of the pH variation in governing ERp44-TMR activity in vivo. A deeper knowledge of the structure/function relationship of ERp44 will shed light on the protein quality control mechanisms thus providing essential knowledge of ESC processing diseases and in biotechnology, improving the production of man-made therapeutic proteins

The hyperinflammatory spectrum: from defects in cytotoxicity to cytokine control

Cytotoxic lymphocytes kill target cells through polarized release of the content of cytotoxic granules towards the target cell. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytosis (HLH) that occurs in mice and humans with inborn errors of lymphocyte cytotoxic function. The clinical and preclinical data indicate that the damage seen in severe, virally triggered HLH is due to an overwhelming immune system reaction and not the direct effects of the virus per se. The main HLH-disease mechanism, which links impaired cytotoxicity to excessive release of pro-inflammatory cytokines is a prolongation of the synapse time between the cytotoxic effector cell and the target cell, which prompts the former to secrete larger amounts of cytokines (including interferon gamma) that activate macrophages. We and others have identified novel genetic HLH spectrum disorders. In the present update, we position these newly reported molecular causes, including CD48-haploinsufficiency and ZNFX1-deficiency, within the pathogenic pathways that lead to HLH. These genetic defects have consequences on the cellular level on a gradient model ranging from impaired lymphocyte cytotoxicity to intrinsic activation of macrophages and virally infected cells. Altogether, it is clear that target cells and macrophages may play an independent role and are not passive bystanders in the pathogenesis of HLH. Understanding these processes which lead to immune dysregulation may pave the way to novel ideas for medical intervention in HLH and virally triggered hypercytokinemia

Electrophoretic Deposition of WS2 Flakes on Nanoholes Arrays—Role of Used Suspension Medium

Here we optimized the electrophoretic deposition process for the fabrication of WS2 plasmonic nanohole integrated structures. We showed how the conditions used for site-selective deposition influenced the properties of the deposited flakes. In particular, we investigated the effect of different suspension buffers used during the deposition both in the efficiency of the process and in the stability of WS2 flakes, which were deposited on an ordered arrays of plasmonic nanostructures. We observed that a proper buffer can significantly facilitate the deposition process, keeping the material stable with respect to oxidation and contamination. Moreover, the integrated plasmonic structures that can be prepared with this process can be applied to enhanced spectroscopies and for the preparation of 2D nanopores

Anti-reflection coating design for metallic terahertz meta-materials

We demonstrate a silicon-based, single-layer anti-reflection coating that suppresses the reflectivity of metals at near-infrared frequencies, enabling optical probing of nano-scale structures embedded in highly reflective surroundings. Our design does not a ect the interaction of terahertz radiation with metallic structures that can be used to achieve terahertz near-field enhancement. We have verified the functionality of the design by calculating and measuring the reflectivity of both infrared and terahertz radiation from a silicon/gold double layer as a function of the silicon thickness. We have also fabricated the unit cell of a terahertz meta-material, a dipole antenna comprising two 20-nm thick extended gold plates separated by a 2 µm gap, where the terahertz field is locally enhanced. We used the time-domain finite element method to demonstrate that such near-field enhancement is preserved in the presence of the anti-reflection coating. Finally, we performed magneto-optical Kerr e ect measurements on a single 3-nm thick, 1-µm wide magnetic wire placed in the gap of such a dipole antenna. The wire only occupies 2% of the area probed by the laser beam, but its magneto-optical response can be clearly detected. Our design paves the way for ultrafast time-resolved studies, using table-top femtosecond near-infrared lasers, of dynamics in nano-structures driven by strong terahertz radiation

Construction of a Suite of Computable Biological Network Models Focused on Mucociliary Clearance in the Respiratory Tract

Mucociliary clearance (MCC), considered as a collaboration of mucus secreted from goblet cells, the airway surface liquid layer, and the beating of cilia of ciliated cells, is the airways’ defense system against airborne contaminants. Because the process is well described at the molecular level, we gathered the available information into a suite of comprehensive causal biological network (CBN) models. The suite consists of three independent models that represent (1) cilium assembly, (2) ciliary beating, and (3) goblet cell hyperplasia/metaplasia and that were built in the Biological Expression Language, which is both human-readable and computable. The network analysis of highly connected nodes and pathways demonstrated that the relevant biology was captured in the MCC models. We also show the scoring of transcriptomic data onto these network models and demonstrate that the models capture the perturbation in each dataset accurately. This work is a continuation of our approach to use computational biological network models and mathematical algorithms that allow for the interpretation of high-throughput molecular datasets in the context of known biology. The MCC network model suite can be a valuable tool in personalized medicine to further understand heterogeneity and individual drug responses in complex respiratory diseases

Site-Selective Integration of MoS2 Flakes on Nanopores by Means of Electrophoretic Deposition

Here, we propose an easy method for site-selective deposition of two-dimensional (2D) material flakes onto nanoholes by means of electrophoretic deposition. This method can be applied to both simple flat nanostructures and complex three-dimensional structures incorporating nano- holes. The deposition method is here used for the decoration of large ordered arrays of plasmonic structures with either a single or few layers of MoS2 . In principle, the plasmonic field generated by the nanohole can significantly interact with the 2D layer leading to enhanced light−material interaction. This makes our platform an ideal system for hybrid 2D material/ plasmonic investigations. The engineered deposition of 2D materials on plasmonic nanostructures is useful for several important applications such as enhanced light emission, strong coupling, hot-electron generation, and 2D material sensors. Site-selective integration of MoS2 flakes on nanopores by means of electrophoretic deposition