Sang-Hwa Urm

Analysis of Biologically Equivalent Dose of Stereotactic Body Radiotherapy for Primary and Metastatic Lung Tumors

Purpose The purpose of this study was to determine the optimal biologically equivalent dose (BED) for stereotactic body radiotherapy (SBRT) by comparing local control rates in proportion to various total doses and fractionation schedules. Materials and Methods Thirty-four patients with early non-small-cell lung cancer and a single metastatic lung tumor were included in this study. Differences in local control rates were evaluated according to gender, primary tumor site, response, tumor size, and BED. For comparison of BEDs, the prescribed dose for SBRT was stratified according to three groups: high (BED > 146 Gy), medium to high (BED, 106 to 146 Gy), and low to medium (BED < 106 Gy). Results For all patients, the overall local control rate was 85.3% at two years after treatment. Five local recurrences were observed, and, notably, all of them were observed in the low to medium BED group. Significantly higher local control rates were observed for patients with a complete response than for those with a partial response or stable disease (p < 0.001). Twenty-six patients with a tumor size of < 3 cm showed no dose-response relationship in the low to medium, medium to high, and high BED groups, whereas eight patients with a tumor size of ≥ 3 cm showed a significant dose-response relationship. The observed 2-year local recurrence-free survival rates in patients with a tumor size of < 3 cm and in those with a tumor size of ≥ 3 cm were 96.2% and 50.0%, respectively, which were significantly different (p=0.007). Conclusion BED > 100 Gy is required in order to achieve a > 85% local control rate regardless of tumor size. The optimal dose for small tumors of < 3 cm appears to be within a range below 150 Gy BED. Escalation of BED to high levels (> 150 Gy) may be required for patients with a tumor size larger than 3 cm.

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.

Muskelin Interacts with Multi-PDZ Domain Protein 1 (MUPP1) through the PDZ Domain

Protein-protein interactions have a critical role in the regulation of many cellular functions. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain is one of domains that mediate protein-protein interactions. PDZ domains typically bind to the specific motif at the carboxyl (C)-terminal end of partner proteins. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, serves a scaffolding function for structure proteins and signaling proteins, but the cellular function of MUPP1 has not been fully elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and muskelin. Muskelin was recently identified as a GABA A receptor (GABA A R) α1 subunit binding protein and known to have a role in receptor endocytosis and degradation. Muskelin bound to the 3 rd PDZ domain, but not to other PDZ domains of MUPP1. The C-terminal end of muskelin was essential for the interaction with MUPP1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, muskelin but not the C-terminal deleted muskelin was co-immunoprecipitated with MUPP1. In addition, MUPP1 co-localized with muskelin at the same subcellular region in cells. These findings collectively suggest that MUPP1 or its interacting proteins could modulate GABAAR trafficking and turnover through the interaction with muskelin.

Parkin Interacts with the PDZ Domain of Multi-PDZ Domain Protein MUPP1

The localization to specific subcellular sites and the regulation of cell surface receptors and channels are crucial for proper functioning. Postsynaptic density-95/Disks large/Zonula occludens-1 (PDZ)-domain is involved in recognition of and interaction between various proteins, by which the localization and the regulation are mediated. Multi-PDZ domain protein 1 (MUPP1) contains 13 PDZ domains. MUPP1 serves a scaffolding function for structure proteins and signaling proteins, but the mechanism how MUPP1 is stabilized and signalized has not yet been elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and Parkin. Parkin is an E3 ubiquitin ligase. Loss-of-function mutations of Parkin gene are known to cause an autosomal recessive juvenile parkinsonism. Parkin bound to the 12th PDZ domain, but not to other PDZ domains of MUPP1. The C-terminal end of Parkin has a type II PDZ-association motif, which was essential for the interaction with MUPP1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, Parkin co-localized with MUPP1. When co-expressed with ubiquitin in HEK-293T cells, MUPP1 has been strongly ubiquitinated by Parkin. These findings collectively suggest that MUPP1 is a novel substrate of Parkin and its function or stability could be modulated by Parkin-mediated ubiquitination.

Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains

Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.

Ferritin, an Iron Storage Protein, Associates with Kinesin 1 through the Cargo-binding Region of Kinesin Heavy Chains (KHCs)

The intracellular transport of organelles and protein complexes is mediated by kinesin superfamily proteins (KIFs). The first kinesin, kinesin 1, was identified as a molecular motor protein that moves various organelles and protein complexes along the microtubule rails in cells. Kinesin 1 is a tetramer of two heavy chains (KHCs, also called KIF5s) and two kinesin light chains (KLCs). KIF5s interact with many different proteins through their tail region, but their binding proteins have not yet been fully identified. To identify the interaction proteins for KIF5A, we performed yeast two-hybrid screening and found a specific interaction with ferritin heavy chain (Frt-h), which has a role in iron storage and detoxification. Frt-h bound to the amino acid residues between 800 and 940 of KIF5A and to other KIF5s in the yeast two-hybrid assay. The coiled-coil domain of Frt-h is essential for interaction with KIF5A. In addition, ferritin light chain (Frt-l) interacted with KIF5s in the yeast two-hybrid assay. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to KHC specifically co-immunoprecipitated Frt-h and Frt-l from mouse brain extracts. These results suggest the kinesin 1 motor protein may transport the ferritin complex in cells.

Wdpcp, a Protein that Regulates Planar Cell Polarity, Interacts with Multi-PDZ Domain Protein 1 (MUPP1) through a PDZ Interaction

Protein-protein interactions regulate the subcellular localization and function of receptors, enzymes, and cytoskeletal proteins. Proteins containing the postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain have potential to act as scaffolding proteins and play a pivotal role in various processes, such as synaptic plasticity, neural guidance, and development, as well as in the pathophysiology of many diseases. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, has a scaffolding function in the clustering of surface receptors, organization of signaling complexes, and coordination of cytoskeletal dynamics. However, the cellular function of MUPP1 has not been fully elucidated. In the present study, a yeast two-hybrid system was used to identify proteins that interacted with the N-terminal PDZ domain of MUPP1. The results revealed an interaction between MUPP1 and Wdpcp (formerly known as Fritz). Wdpcp was identified as a planar cell polarity (PCP) effector, which is known to have a role in collective cell migration and cilia formation. Wdpcp bound to the PDZ1 domain but not to other PDZ domains of MUPP1. The C-terminal end of Wdpcp was essential for the interaction with MUPP1 in the yeast two-hybrid assay. This interaction was further confirmed in a glutathione S-transferase (GST) pull-down assay. When coexpressed in HEK-293T cells, Wdpcp was coimmunoprecipitated with MUPP1. In addition, MUPP1 colocalized with Wdpcp at the same subcellular region in cells. Collectively, these results suggest that the MUPP1-Wdpcp interaction could modulate actin cytoskeleton dynamics and polarized cell migration.

The Scaffolding Protein WAVE1 Associates with Kinesin 1 through the Tetratricopeptide Repeat (TPR) Domain of the Kinesin Light Chain (KLC)

Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motor proteins essential for the intracellular transport of organelles and protein complexes in cells. Kinesin 1 is a member of those KIFs that transport various cargoes, including organelles, synaptic vesicles, neurotransmitter receptors, cell signaling molecules, and mRNAs through interaction between its light chain subunit and the cargoes. Kinesin light chains (KLCs) are non-motor subunits that associate with the kinesin heavy chain (KHC) dimer. KLCs interact with many different binding proteins, but their particular binding proteins have not yet been fully identified. We used the yeast two-hybrid assay to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. We found an interaction between the TPR domain of KLC1 and Wiskott-Aldrich syndrome protein family member 1 (WAVE1), a member of the WASP/WAVE family involved in regulation of actin cytoskeleton. WAVE1 bound to the six TPR domain-containing regions of KLC1 and did not interact with KHCs (KIF5A, KIF5B, and KIF5C) in the yeast two-hybrid assay. The carboxyl (C)-terminal verprolin-cofilin-acidic (VCA) domain of WAVE1 is essential for interaction with KLC1. Also, other WAVE isoforms (WAVE2 and WAVE3) interacted with KLC1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, WAVE1 co-localized with KLC1 and co-immunoprecipitated with KLC1 and KIF5B. These results suggest that kinesin 1 motor protein may transport WAVE complexes or WAVE-coated cargoes in cells.

Cadms/SynCAMs/Necls/TSLCs Interact with Multi-PDZ Domain Protein MUPP1

Cell adhesion molecules determine the cell-cell binding and the interactions between cells and extracellular signals. Cell-cell junctional complexes, which maintain the structural integrity of tissues, consist of more than 50 proteins including multi-PDZ domain protein 1 (MUPP1). MUPP1 contains 13 postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domains and serves a scaffolding function for transmembrane proteins and cytoskeletal proteins or signaling proteins, but the mechanism how MUPP1 links and stabilizes the juxtamembrane proteins has not yet been elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and cell adhesion molecule 1 (Cadm1, also known as SynCAM1, Necl-2, or TSLC1). Cadm1 bound to the second PDZ domain of MUPP1. The carboxyl (C)-terminal end of Cadm1 has a type II PDZ-association motif (-Y-F-I) which was essential for the interaction with MUPP1 in the yeast two-hybrid assay. MUPP1 also bound to the C-terminal cytoplasmic tail region of other Cadm family members (Cadm2, Cadm3, and Cadm4). In addition, these protein-protein interactions were observed in the glutathione S-transferase (GST) pull-down assay and by coimmunoprecipitation. Anti-MUPP1 antibody co-immunoprecipitated Cadm1 and Cadm4 with MUPP1 from mouse brain extracts. These results suggest that MUPP1 could mediate interaction between Cadms and cytoskeletal proteins.