Dae-Hyun Seog

A Novel Motor, KIF13A, Transports Mannose-6-Phosphate Receptor to Plasma Membrane through Direct Interaction with AP-1 Complex

Intracellular transport mediated by kinesin superfamily proteins (KIFs) is a highly regulated process. The molecular mechanism of KIFs binding to their respective cargoes remains unclear. We report that KIF13A is a novel plus end-directed microtubule-dependent motor protein and associates with beta 1-adaptin, a subunit of the AP-1 adaptor complex. The cargo vesicles of KIF13A contained AP-1 and mannnose-6-phosphate receptor (M6PR). Overexpression of KIF13A resulted in mislocalization of the AP-1 and the M6PR. Functional blockade of KIF13A reduced cell surface expression of the M6PR. Thus, KIF13A transports M6PR-containing vesicles and targets the M6PR from TGN to the plasma membrane via direct interaction with the AP-1 adaptor complex.

Molecular Motor Proteins of the Kinesin Superfamily Proteins (KIFs): Structure, Cargo and Disease

Intracellular organelle transport is essential for morphogenesis and functioning of the cell. Kinesins and kinesin-related proteins make up a large superfamily of molecular motors that transport cargoes such as vesicles, organelles (e.g. mitochondria, peroxisomes, lysosomes), protein complexes (e.g. elements of the cytoskeleton, virus particles), and mRNAs in a microtubule- and ATP-dependent manner in neuronal and non-neuronal cells. Until now, more than 45 kinesin superfamily proteins (KIFs) have been identified in the mouse and human genomes. Elucidating the transport pathways mediated by kinesins, the identities of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. This review focuses on the structure, the binding partners of kinesins and kinesin-based human diseases.

Glutamate Receptor-interacting Protein 1 Protein Binds to the Microtubule-associated Protein

To identify the interaction proteins for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.

Interleukin-4 C-590T polymorphism is associated with protection against nasal polyps in a Korean population.

Background Although many studies have implicated interleukin (IL)-4 promoter polymorphisms as potential determinants of disease susceptibility, there are no reports on the association between IL-4 promoter polymorphisms and nasal polyps. The aim of this study was to investigate the relationship between an IL-4 promoter polymorphism and nasal polyps. Methods The C-590T promoter polymorphism of the IL-4 gene was genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis in 106 Korean chronic rhinosinusitis patients with or without nasal polyps and 70 healthy Korean subjects. Results The frequency of the T allele at position -590 of the IL-4 gene in a Korean population was 0.85, which was significantly higher than those of other ethnic groups, and the T/T allele at position -590 of IL-4 was associated with protection against nasal polyps if compared with the C/C allele (relative risk, 0.529; 95% confidence interval, 0.307–0.912; p = 0.028). Conclusion T-590, which is dominant at position -590 of the IL-4 promoter, appears to be associated with a protective mechanism against nasal polyps in Korean populations.

Transmembrane and Ubiquitin-Like Domain-Containing Protein 1 (Tmub1/HOPS) Facilitates Surface Expression of GluR2-Containing AMPA Receptors

Some ubiquitin-like (UBL) domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported. Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS) protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP) were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

Sorting Nexin 17 Interacts Directly with Kinesin Superfamily KIF1Bβ Protein

KIF1Bbeta is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1Bbeta is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1Bbeta is still unclear. To elucidate the KIF1Bbeta-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1Bbeta and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1Bbeta. SNX17 protein bound to the specific region of KIF1Bbeta (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1Bbeta associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1Bbeta-mediated transport as a KIF1Bbeta adaptor protein.

Protrusion of N-acetylglucosamine Kinase Clusters into the Base of Excitatory Synapses

N-Acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) catalyzes the phosphorylation of GlcNAc to GlcNAc-6-phosphate (GlcNAc-6-P). Despite detailed characterization of the enzyme itself, there have been few studies on the expression of NAGK in mammalian tissues. In the rat hippocampal neuron in culture, NAGK-immunoreactivity (IR) formed clusters in somatodendritic domains. In this study we characterized the NAGK clusters that protrude out the long axis of dendritic shafts. By double-labeling of the neurons with antibodies against NAGK and various synaptic proteins, we show that NAGK is positioned at the base of spines, while there were no NAGK protrusions into inhibitory postsynaptic sites. Immunoblot analysis showed that NAGK was included in synaptosomes but not in PSD fractions. Our results indicate that the NAGK clusters at the dendritic periphery protrude into spines.

N-Acetyl-D-Glucosamine Kinase Is a Component of Nuclear Speckles and Paraspeckles

Protein O-GlcNAcylation, dictated by cellular UDP-N-acetylglucosamine (UDP-GlcNAc) levels, plays a crucial role in posttranslational modifications. The enzyme GlcNAc kinase (NAGK, E.C. 2.7.1.59) catalyzes the formation of GlcNAc-6-phosphate, which is a major substrate for the biosynthesis of UDP-GlcNAc. Recent studies have revealed the expression of NAGK in different types of cells especially in neuronal dendrites. Here, by immunocytochemistry (ICC) and immunonucleochemistry (INC) of cultured rat hippocampal neurons, HEK293T and GT1-7 cells, we have showed that NAGK immuno-reactive punctae being present in the nucleoplasm colocalized with small nuclear ribonucleoprotein-associated protein N (snRNPN) and p54NRB, which are speckle and paraspeckle markers, respectively. Furthermore, NAGK IR cluster was also found to be colocalized with GTF2H5 (general transcription factor IIH, polypeptide 5) immuno reactive punctae. In addition, relative localization to the ring of nuclear lamin matrix and to GlcNAc, which is highly enriched in nuclear pore complexes, showed that NAGK surrounds the nucleus at the cytoplasmic face of the nuclear outer membrane. By in situ proximity ligation assay (PLA) we confirmed the colocalization of NAGK with snRNPN in the nucleus and in dendrites, while we also verified the interactions of NAGK with p54NRB, and with GTF2H5 in the nucleus. These associations between NAGK with speckle, paraspeckle and general transcription factor suggest its regulatory roles in gene expression.

PDZ Domain-containing Proteins at Autotypic Junctions in Myelinating Schwann Cells

A type of cell junction that is formed between different parts within the same cell is called autotypic cell junction. Autotypic junction proteins form tight junctions found between membrane lamellae of a cell, especially in myelinating glial cells. Some of them have postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domains, which interact with the carboxyl (C)-terminal PDZ-binding motif of other proteins. PDZ domains are protein-protein interaction modules that play a role in protein complex assembly. The PDZ domain, which is widespread in bacteria, plants, yeast, metazoans, and Drosophila, allows the assembly of large multi-protein complexes. The multi-protein complexes act in intracellular signal transduction, protein targeting, and membrane polarization. The identified PDZ domain-containing proteins located at autotypic junctions include zonula occludens-1 (ZO-1), ZO-2, pals-1-associated tight junction protein (PATJ), multi-PDZ domain proteins (MUPPs), membrane-associated guanylate kinase inverted 2 (MAGI2), and protease-activated receptor (PAR)-3. PAR-3 interacts with atypical protein kinase C and PAR-6, forming a ternary complex, which plays an important role in the regulation of cell polarity. MAGI2 interacts with α-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) receptor at excitatory synapses. PATJ is detected in paranodal loops associated with claudin-1. On the other hand, MUPP1 is found in mesaxons and Schmidt-Lanterman incisures with claudin-5. ZO-1, ZO-2, and PAR-3 are found at all three sites. Different distributions of PDZ domain-containing proteins affect the development of autotypic junctions. In this review, we will describe PDZ domain-containing proteins at autotypic tight junctions in myelinating Schwann cells and their roles.

Expressed Sequence Tags (ESTs) analysis of Tenebrio molitor larvae

Tenebrio molitor has been seriously investigated as a model insect in elucidating Toll signaling pathway and related regulators of innate immunity. However, little is known with regards to the genomic information in T. molitor. In an attempt towards exploiting the rich transcriptomics data that would offer further insights into the study on insect immunity through potential screening of immune‐related genes in the model insect, we constructed a cDNA library (library titer = 5.0 × 105pfu/ml) of T. molitor larvae and analyzed expressed sequence tag (EST) sequences from 1056 clones. The base calling and quality check of obtained chromatogram files were performed by using the Phred program (trim_alt 0.05 (P‐score > 20). After removal of vector and 100 bp and less sequences, 1023 sequences were generated having an average insert size of 792 bp, including 160 clusters, 164 contigs and 387 singletons through clustering and assembling process using the TGI Clustering Tools (TGICL) package. The unique EST sequences were searched against the NCBI nr database by local BLAST (blastx, E < e−5) with 940 sequences showing significant hits. Subsequently, KOG (clusters of orthologous groups for eukaryotic complete genomes) analysis was conducted (blastx, E < e−10) towards prediction of transcriptomal functions, leading to the categorization of 638 genes. The majority of genes belonged to Z category (cytoskeleton‐related genes), R category (general function prediction), and C category (energy production and conversion related genes). These basic EST datasets and their bioinformatics analysis will be helpful in investigating the biological mechanism and molecular pathway related genes involved in innate immunity mechanisms of T. molitor.