Shunsuke Kita

Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

ABSTRACT Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection.

Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

Emerging evidence has revealed the pivotal roles of C‐type lectin‐like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor‐P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin‐like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A‐LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor‐binding face of the C‐type lectin‐like domain is flat, and forms an extended β‐sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte‐associated C‐type lectin, which binds to the CTLR member, NKp65. A LLT1‐NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte‐associated C‐type lectin‐NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.

Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid-protein interactions in KRN7000/CD1d complexes.

Establishment of the BacMam system using silkworm baculovirus.

The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate-buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy.

X‐ray crystal structure of Escherichia coli HspQ, a protein involved in the retardation of replication initiation

The heat shock protein HspQ (YccV) of Escherichia coli has been proposed to participate in the retardation of replication initiation in cells with the dnaA508 allele. In this study, we have determined the 2.5‐Å‐resolution X‐ray structure of the trimer of HspQ, which is also the first structure of a member of the YccV superfamily. The acidic character of the HspQ trimer suggests an interaction surface with basic proteins. From these results, we discuss the cellular function of HspQ, including its relationship with the DnaA508 protein.

Structures and Functions of MHC-like Proteins

Classical MHC molecules utilize their α1-α2 domains to display endogenous (self and nonself) peptides for antigen presentation to immune cells. However, over the past few decades many investigations have revealed a wide range of structural and functional variations of MHC superfamily members. Here we focus on the MHC-like proteins harboring distinct functions from those of the classical MHCs, such as Fc binding to transport IgG (FcRn), fatty acid homeostasis (ZAG), Fe ion metabolism (HFE), other immune responses (T proteins, M proteins, and ULBPs), and immune evasion of viral infection (UL18, UL142, m138, m144, m145, m152, m155, m157, etc.). These MHC-like proteins have basically lost the ability to present peptides, due to modulations of the typical antigen-binding groove. Instead, the surfaces have become receptor recognition sites. We further highlight the various tactics employed to elegantly control the physiological functions by using expanded and diversified protein surfaces, such as those generated by domain deletion and heavy sugar modification.

Structural Aspects of C-Type Lectin Receptors

Numerous structural analyses (X-ray crystallography and NMR) of C-type lectin receptors (CLRs) have been performed, because CLRs are not only attractive as important molecules in immunity and infectious diseases but also as drug targets. In CLRs, high amino acid sequence similarity exists in the extracellular carbohydrate recognition domains (CRDs), which are responsible for ligand binding. However, recent functional analyses of CLRs implied that these molecules recognize a wide variety of ligands in addition to saccharides, including glycopeptides, glycolipids, and proteins. In this chapter, we focus on structural studies of CLRs. We first summarize the structural features conserved among the CRDs and then describe how each C-type lectin receptor elegantly achieves its distinct ligand specificity, by illustrating the structural aspects of several representative CLRs.