Fever Promotes T Lymphocyte Trafficking via a Thermal Sensory Pathway Involving Heat Shock Protein 90 and α4 Integrins.

Fever is an evolutionarily conserved response that confers survival benefits during infection. However, the underlying mechanism remains obscure. Here, we report that fever promoted T lymphocyte trafficking through heat shock protein 90 (Hsp90)-induced α4 integrin activation and signaling in T cells. By inducing selective binding of Hsp90 to α4 integrins, but not ß2 integrins, fever increased α4-integrin-mediated T cell adhesion and transmigration. Mechanistically, Hsp90 bound to the α4 tail and activated α4 integrins via inside-out signaling. Moreover, the N and C termini of one Hsp90 molecule simultaneously bound to two α4 tails, leading to dimerization and clustering of α4 integrins on the cell membrane and subsequent activation of the FAK-RhoA pathway. Abolishment of Hsp90-α4 interaction inhibited fever-induced T cell trafficking to draining lymph nodes and impaired the clearance of bacterial infection. Our findings identify the Hsp90-α4-integrin axis as a thermal sensory pathway that promotes T lymphocyte trafficking and enhances immune surveillance during infection.

Interaction of human dendritic cell receptor DEC205/CD205 with keratins.

DEC205 (CD205) is one of the major endocytic receptors on dendritic cells and has been widely used as a receptor target in immune therapies. It has been shown that DEC205 can recognize dead cells through keratins in a pH-dependent manner. However, the mechanism underlying the interaction between DEC205 and keratins remains unclear. Here we determine the crystal structures of an N-terminal fragment of human DEC205 (CysR∼CTLD3). The structural data show that DEC205 shares similar overall features with the other mannose receptor family members such as the mannose receptor and Endo180, but the individual domains of DEC205 in the crystal structure exhibit distinct structural features that may lead to specific ligand binding properties of the molecule. Among them, CTLD3 of DEC205 adopts a unique fold of CTLD, which may correlate with the binding of keratins. Furthermore, we examine the interaction of DEC205 with keratins by mutagenesis and biochemical assays based on the structural information and identify an XGGGX motif on keratins that can be recognized by DEC205, thereby providing insights into the interaction between DEC205 and keratins. Overall, these findings not only improve the understanding of the diverse ligand specificities of the mannose receptor family members at the molecular level but may also give clues for the interactions of keratins with their binding partners in the corresponding pathways.

Assembly and recognition of keratins: A structural perspective.

Keratins are one of the major components of cytoskeletal network and assemble into fibrous structures named intermediate filaments (IFs), which are important for maintaining the mechanical properties of cells and tissues. Over the past decades, evidence has shown that the functions of keratins go beyond providing mechanical support for cells, they interact with multiple cellular components and are widely involved in the pathways of cell proliferation, differentiation, motility and death. However, the structural details of keratins and IFs are largely missing and many questions remain regarding the mechanisms of keratin assembly and recognition. Here we briefly review the current structural models and assembly of keratins as well as the interactions of keratins with the binding partners, which may provide a structural view for understanding the mechanisms of keratins in the biological activities and the related diseases.

Structure of cell-cell adhesion mediated by the Down syndrome cell adhesion molecule.

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.

Glycometabolism regulates hepatitis C virus release.

HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response.

On the contact electrification mechanism in semiconductor-semiconductor case by vertical contact-separation triboelectric nanogenerator.

With the speed of industrialization accelerating, the traditional energy is in the predicament of being exhausted. Humans urgently need a clean energy to maintain the peace and development. Triboelectric nanogenerator (TENG) is a tiny device that collects and converts the renewable energy, such as wind, vibration and tidal/blue energy, into electrical energy. As the most significant working principle of TENG, contact electrification (CE) has been broadly studied since it was documented thousands of years ago. A large number of related researches are reported. However, most of them are focused on the polymer materials, device structures and potential applications. There are few literatures about the mechanism of CE, especially in the semiconductor-semiconductor case. Semiconductor-semiconductor CE is a promising method to generate electricity, which has been used in many fields, such as the photodetector and displacement sensor. Therefore, it is necessary to establish a serious and detailed theory in order to deeply explain the underlying mechanisms of semiconductor-semiconductor CE. In this work, a novel Fermi level model based on energy band theory is proposed to illustrate the semiconductor-semiconductor CE mechanism. By assembling a ZnO/Si vertical contact-separation (CS) mode TENG, the charge transfer introduced by CE is systematically measured. According to the energy band theory and TENG governing equation, the experimental data is qualitatively and quantitatively analyzed. Moreover, the effects of different concentrations of growth solutions on the morphology of ZnO nanowires and the Fermi level difference between ZnO and Si are explored as well. Results show that it is the Fermi level difference that dominates the short circuit transfer charge amount and direction of semiconductor-semiconductor CE mechanism. Our work can be applied to understand the CE mechanism in semiconductor-semiconductor case and broaden the application prospects of semiconductor-based TENG.

Recognition of lipoproteins by scavenger receptor class A members.

Scavenger receptor class A (SR-A) proteins are type II transmembrane glycoproteins that form homotrimers on the cell surface. This family has five known members (SCARA1 to 5, or SR-A1 to A5) that recognize a variety of ligands and are involved in multiple biological pathways. Previous reports have shown that some SR-A family members can bind modified low-density lipoproteins (LDLs); however, the mechanisms of the interactions between the SR-A members and these lipoproteins are not fully understood. Here, we systematically characterize the recognition of SR-A receptors with lipoproteins and report that SCARA1 (SR-A1, CD204), MARCO (SCARA2), and SCARA5 recognize acetylated or oxidized LDL and very-low-density lipoprotein in a Ca2+-dependent manner through their C-terminal scavenger receptor cysteine-rich (SRCR) domains. These interactions occur specifically between the SRCR domains and the modified apolipoprotein B component of the lipoproteins, suggesting that they might share a similar mechanism for lipoprotein recognition. Meanwhile, SCARA4, a SR-A member with a carbohydrate recognition domain instead of the SRCR domain at the C terminus, shows low affinity for modified LDL and very-low-density lipoprotein but binds in a Ca2+-independent manner. SCARA3, which does not have a globular domain at the C terminus, was found to have no detectable binding with these lipoproteins. Taken together, these results provide mechanistic insights into the interactions between SR-A family members and lipoproteins that may help us understand the roles of SR-A receptors in lipid transport and related diseases such as atherosclerosis.

Interactions of ferritin with scavenger receptor class A members.

Scavenger receptors are a superfamily of membrane-bound receptors that recognize both self and nonself targets. Scavenger receptor class A (SR-A) has five known members (SCARA1 to -5 or SR-A1 to -A5), which are type II transmembrane proteins that form homotrimers on the cell surface. SR-A members recognize various ligands and are involved in multiple biological pathways. Among them, SCARA5 can function as a ferritin receptor; however, the interaction between SCARA5 and ferritin has not been fully characterized. Here, we determine the crystal structures of the C-terminal scavenger receptor cysteine-rich (SRCR) domain of both human and mouse SCARA5 at 1.7 and 2.5 Å resolution, respectively, revealing three Ca2+-binding sites on the surface. Using biochemical assays, we show that the SRCR domain of SCARA5 recognizes ferritin in a Ca2+-dependent manner, and both L- and H-ferritin can be recognized by SCARA5 through the SRCR domain. Furthermore, the potential binding region of SCARA5 on the surface of ferritin is explored by mutagenesis studies. We also examine the interactions of ferritin with other SR-A members and find that SCARA1 (SR-A1, CD204) and MARCO (SR-A2, SCARA2), which are highly expressed on macrophages, also interact with ferritin. By contrast, SCARA3 and SCARA4, the two SR-A members without the SRCR domain, have no detectable binding with ferritin. Overall, these results provide a mechanistic view regarding the interactions between the SR-A members and ferritin that may help to understand the regulation of ferritin homeostasis by scavenger receptors.

Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus.

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.

Architecture of cell-cell adhesion mediated by sidekicks.

Cell-cell adhesion is important for cell growth, tissue development, and neural network formation. Structures of cell adhesion molecules have been widely studied by crystallography, revealing the molecular details of adhesion interfaces. However, due to technical limitations, the overall structure and organization of adhesion molecules at cell adhesion interfaces has not been fully investigated. Here, we combine electron microscopy and other biophysical methods to characterize the structure of cell-cell adhesion mediated by the cell adhesion molecule Sidekick (Sidekick-1 and Sidekick-2) and obtain 3D views of the Sidekick-mediated adhesion interfaces as well as the organization of Sidekick molecules between cell membranes by electron tomography. The results suggest that the Ig-like domains and the fibronectin III (FnIII) domains of Sidekicks play different roles in cell adhesion. The Ig-like domains mediate the homophilic transinteractions bridging adjacent cells, while the FnIII domains interact with membranes, resulting in a tight adhesion interface between cells that may contribute to the specificity and plasticity of cell-cell contacts during cell growth and neural development.

A Nanoparticle-Based Hepatitis C Virus Vaccine With Enhanced Potency.

Despite the emergence of new direct-acting antivirals, hepatitis C virus (HCV) chronic infection and its consequent fibrosis and hepatocarcinoma remain a significant burden for public health, thus requiring an effective preventive vaccine. Our group previously showed that a subunit vaccine based on recombinant soluble E2 (sE2) can induce broadly neutralizing antibodies. To improve the immunogenicity of sE2, we designed and produced a fusion protein (sE2-ferritin) comprising sE2 and a ferritin unit in Drosophila S2 cells, which self-assembled into a nanoparticle with sE2 displayed on the surface. The sE2 moiety on the sE2-ferritin nanoparticle not only had nearly natural conformation but also had better affinities than the unfused sE2 to neutralizing antibodies, receptor, and patient serum. Mouse immunization studies showed that sE2-ferritin was more potent than sE2 in inducing anti-HCV broadly neutralizing antibodies. Our results demonstrate that sE2-ferritin is a vaccine candidate superior to previously developed sE2, providing a new possibility for controlling HCV.

The scavenger receptor SCARA1 (CD204) recognizes dead cells through spectrin.

Scavenger receptor class A member 1 (SCARA1 or CD204) is an immune receptor highly expressed on macrophages. It forms homotrimers on the cell surface and plays important roles in regulating immune responses via its involvement in multiple pathways. However, both the structure and the functional roles of SCARA1 are not fully understood. Here, we determined the crystal structure of the C-terminal SRCR domain of SCARA1 at 1.8 Å resolution, revealing its Ca2+-binding site. Results from cell-based assays revealed that SCARA1 can recognize dead cells, rather than live cells, specifically through its SRCR domain and in a Ca2+-dependent manner. Furthermore, by combining MS and biochemical assays, we found that cellular spectrin is the binding target of SCARA1 on dead cells and that the SRCR domain of SCARA1 recognizes the SPEC repeats of spectrin in the presence of Ca2+ We also found that macrophages can internalize dead cells or debris from both erythrocytes and other cells through the interaction between SCARA1 and spectrin, suggesting that SCARA1 could function as a scavenging receptor that recognizes dead cells. These results suggest that spectrin, which is one of the major components of the cytoskeleton, acts as a cellular marker that enables the recognition of dead cells by the immune system.

Silicon nanowire core-shell PN junction phototransistors by self-assembled monolayer doping.

Nanoscale photoconductors often have extremely high gain in quantum efficiency but suffer from the difficulty to design the density of surface states that cause the high photogain. In this Letter, we created high-gain photoconductors by forming a core-shell PN junction in silicon nanowires via self-assembled molecular monolayer doping. The highly doped n-type shell deactivates all the surface states by filling with electrons so that the n-type shell as a well, instead of the surface states, captures and emits photogenerated minority electrons under ON/OFF light illumination. The corresponding excess majority holes are accumulated in the nanowire channel and thus modulate the channel width, resulting in the experimentally observed high photogain (∼108). The photoresponses of these phototransistors were systematically investigated as a function of the nanowire width and light illumination intensity. The results show that the nanowire channel is pinched off for the nanowires narrower than 73 nm due to the core-shell PN junction. We further derived analytical equations based on the PN junction device principle, finding the explicit gain equation that governs the photogain as a function of light intensity and other physical parameters of the nanowires. The explicit gain equations can fit well with the experimental data and allow us to design the core-shell nanowire phototransitors with desired performance.

Crystal structure of the CTLD7 domain of human M-type phospholipase A2 receptor.

M-type phospholipase A2 receptor (PLA2R) is a member of the mannose receptor family. Recent evidence shows that PLA2R is a major autoantigen causing idiopathic membranous nephropathy (IMN), which is an autoimmune disease and one of the most common causes for nephrotic syndrome in adults. The epitope mapping data suggest that the major epitopes of PLA2R locate at the CysR, CTLD1 and CTLD7 domains. However, due to the lack of the high-resolution structural information, it is unclear how the autoantibodies interact with PLA2R. Here we determine the crystal structure of the CTLD7 domain of PLA2R at 1.8 Å, showing that it adopts a typical CTLD fold, and the structural alignments also provide hints for the potential antibody binding regions. In addition, the high-resolution structural information of CTLD7 could be applied to identify the epitopes for autoantibodies, which would facilitate the therapeutic strategies against IMN.

Structural basis of the pH-dependent conformational change of the N-terminal region of human mannose receptor/CD206.

Mannose receptor (MR, CD206) is an immune receptor highly expressed on macrophages and plays important roles in glycoprotein clearance, immune response and matrix turnover. Previous studies have shown that MR recognizes multiple ligands and recycles between cell surface and endosomes, and the conformation and ligand binding of MR are regulated by environmental pH. However, due to the lack of high-resolution details, the mechanisms of the pH-dependent properties of MR have not been fully understood. Here we investigate the pH-dependent conformational change of MR by solving a series of crystal structures of MR N-terminal fragments (CysR~CTLD2/3) at pH ranging from 4.0 to 8.5. The results show that the CTLD3 domain plays a critical role in regulating the conformational change of the N-terminal region of MR by forming interactions with the CTLD2 domain specifically at acidic pH. Moreover, the structural data also show the conformational changes of the 4-SO4-GalNAc binding pocket at the CysR domain, which might be relevant to the binding and release of the ligand. Overall, these results provide a model for the pH-dependent conformational change of the N-terminal region of MR that may help to understand its functional mechanism at molecular level.

Syntenin regulates hepatitis C virus sensitivity to neutralizing antibody by promoting E2 secretion through exosomes.

BACKGROUND & AIMS: Assembly of infectious hepatitis C virus (HCV) particles is known to involve host lipoproteins, giving rise to unique lipo-viro-particles (LVPs), but proteome studies now suggest that additional cellular proteins are associated with HCV virions or other particles containing the viral envelope glycoprotein E2. Many of these host cell proteins are common markers of exosomes, most notably the intracellular adaptor protein syntenin, which is required for exosome biogenesis. We aimed to elucidate the role of syntenin/E2 in HCV infection. METHODS: Using cell culture-derived HCV, we studied the biogenesis and function of E2-coated exosomes in both hepatoma cells and primary human hepatocytes (PHHs). RESULTS: Knockout of syntenin had a negligible impact on HCV replication and virus production, whereas ectopic expression of syntenin at physiological levels reduced intracellular E2 abundance, while concomitantly increasing the secretion of E2-coated exosomes. Importantly, cells expressing syntenin and HCV structural proteins efficiently released exosomes containing E2 but lacking the core protein. Furthermore, infectivity of HCV released from syntenin-expressing hepatoma cells and PHHs was more resistant to neutralization by E2-specific antibodies and chronic-phase patient serum. We also found that high E2/syntenin levels in sera correlate with lower serum neutralization capability. CONCLUSIONS: E2- and syntenin-containing exosomes are a major type of particle released from cells expressing high levels of syntenin. Efficient production of E2-coated exosomes renders HCV infectivity less susceptible to antibody neutralization in hepatoma cells and PHHs. LAY SUMMARY: This study identifies a key role for syntenin in the regulation of E2 secretion via exosomes. Efficient production of E2-coated exosomes was shown to make hepatitis C virus less sensitive to antibody neutralization. These results may have implications for the development of a hepatitis C virus vaccine.

Keratin mediates the recognition of apoptotic and necrotic cells through dendritic cell receptor DEC205/CD205.

Clearance of dead cells is critical for maintaining homeostasis and prevents autoimmunity and inflammation. When cells undergo apoptosis and necrosis, specific markers are exposed and recognized by the receptors on phagocytes. DEC205 (CD205) is an endocytotic receptor on dendritic cells with antigen presentation function and has been widely used in immune therapies for vaccine generation. It has been shown that human DEC205 recognizes apoptotic and necrotic cells in a pH-dependent fashion. However, the natural ligand(s) of DEC205 remains unknown. Here we find that keratins are the cellular ligands of human DEC205. DEC205 binds to keratins specifically at acidic, but not basic, pH through its N-terminal domains. Keratins form intermediate filaments and are important for maintaining the strength of cells and tissues. Our results suggest that keratins also function as cell markers of apoptotic and necrotic cells and mediate a pH-dependent pathway for the immune recognition of dead cells.

pH-Dependent recognition of apoptotic and necrotic cells by the human dendritic cell receptor DEC205.

Dendritic cells play important roles in regulating innate and adaptive immune responses. DEC205 (CD205) is one of the major endocytotic receptors on dendritic cells and has been widely used for vaccine generation against viruses and tumors. However, little is known about its structure and functional mechanism. Here we determine the structure of the human DEC205 ectodomain by cryoelectron microscopy. The structure shows that the 12 extracellular domains form a compact double ring-shaped conformation at acidic pH and become extended at basic pH. Biochemical data indicate that the pH-dependent conformational change of DEC205 is correlated with ligand binding and release. DEC205 only binds to apoptotic and necrotic cells at acidic pH, whereas live cells cannot be recognized by DEC205 at either acidic or basic conditions. These results suggest that DEC205 is an immune receptor that recognizes apoptotic and necrotic cells specifically through a pH-dependent mechanism.

Yeast Hmt1 catalyses asymmetric dimethylation of histone H3 arginine 2 in vitro.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate protein arginine residues. PRMTs' substrates include histones and a variety of non-histone proteins. Previous studies have shown that yeast Hmt1 is a type I PRMT and methylates histone H4 arginine 3 and several mRNA-binding proteins. Hmt1 forms dimers or oligomers, but how dimerization or oligomerization affects its activity remains largely unknown. We now report that Hmt1 can methylate histone H3 arginine 2 (H3R2) in vitro. The dimerization but not hexamerization is essential for Hmt1's activity. Interestingly, the methyltransferase activity of Hmt1 on histone H3R2 requires reciprocal contributions from two Hmt1 molecules. Our results suggest an intermolecular trans-complementary mechanism by which Hmt1 dimer methylates its substrates.

Structure of FcRY, an avian immunoglobulin receptor related to mammalian mannose receptors, and its complex with IgY.

Fc receptors transport maternal antibodies across epithelial cell barriers to passively immunize newborns. FcRY, the functional counterpart of mammalian FcRn (a major histocompatibility complex homolog), transfers IgY across the avian yolk sac, and represents a new class of Fc receptor related to the mammalian mannose receptor family. FcRY and FcRn bind immunoglobulins at pH ≤6.5, but not pH ≥7, allowing receptor-ligand association inside intracellular vesicles and release at the pH of blood. We obtained structures of monomeric and dimeric FcRY and an FcRY-IgY complex and explored FcRY's pH-dependent binding mechanism using electron cryomicroscopy (cryoEM) and small-angle X-ray scattering. The cryoEM structure of FcRY at pH 6 revealed a compact double-ring "head," in which the N-terminal cysteine-rich and fibronectin II domains were folded back to contact C-type lectin-like domains 1-6, and a "tail" comprising C-type lectin-like domains 7-8. Conformational changes at pH 8 created a more elongated structure that cannot bind IgY. CryoEM reconstruction of FcRY dimers at pH 6 and small-angle X-ray scattering analysis at both pH values confirmed both structures. The cryoEM structure of the FcRY-IgY revealed symmetric binding of two FcRY heads to the dimeric FcY, each head contacting the C(H)4 domain of one FcY chain. FcRY shares structural properties with mannose receptor family members, including a head and tail domain organization, multimerization that may regulate ligand binding, and pH-dependent conformational changes. Our results facilitate understanding of immune recognition by the structurally related mannose receptor family and comparison of diverse methods of Ig transport across evolution.