신경세포의 발달에서의 Cullin-RING E3 유비퀴틴-리가아제 4에 대한 연구

Neuritogenesis, CRL4, axonal morphogenesis, neurons, cullin proteinsI. Introduction 1 1.1 General principle of neuronal development 1 1.2 The role of ubiquitination during neurogenesis 11 1.3 The role of Cullins during neurogenesis 14 II. Aims of the project 18 2.1 When and Where Cullin 4 proteins are expressed and active during neuronal development 18 2.2 How are the level and activity of Cul4 proteins regulated 18 2.3 What are the functions of the CRL4 complex during neuronal development 18 2.4 How does the CRL4 function and whom does it work with 18 III. Materials and methods 19 3.1 Plasmids and Antibodies 19 3.2 Experimental animals 21 3.3 Cloning and work with plasmid DNA 21 3.4 RNA isolation and RT-PCRs 21 3.5 Primary neuronal cell cultures and pc12 cell culture 21 3.6 Immunocytochemistry and proximity ligation assay 22 3.7 Immunohistochemistry 23 3.8 Fractionation of nucleus and cytosol 23 3.9 Immunoprecipitation 24 3.10 Immunoblotting 24 3.11 LC-MS/MS 24 3.12 Image acquisition and analysis of neuronal morphology 25 3.13 Statistical analysis 25 IV. Results 26 4.1 Cul4a and Cul4b are up-regulated during early neuronal development 26 4.2 Neuronal Activity inhibits cul4a and cul4b function. 40 4.3 Regulation of the level or activity of Cul4a and Cul4b via genetic and pharmacological tools 46 4.4 Cul4a and Cul4b regulate Axonal outgrowth and branching in early neuronal development 50 4.5 CRL4 interacts with cytoskeleton-organizing proteins 61 4.6 DCX as a promising target of Cul4a and Cul4b 66 V. Discussion 71 VI. Conclusion 75 VII. References 77 VIII. 요 약 문 87DoctordCollectio

Comparison of physicochemically conserved residues of human odorant receptors within two classes

Human ORs included in class A GPCRs are phylogenetically divided into two clusters (Class I and Class II ORs) by their sequence identity. Interestingly, odorants that these classes each respond to seem to have different physicochemical features. Experimentally tested agonists of class I ORs tend to be more hydrophilic than those of class II ORs. Therefore, comparing the amino acid sequences of ORs within the classes may allow us to elucidate the structure-function relationships in ORs further. However, no study applied the physicochemical conservation analysis on the comparison of OR classes, which may provide more information about the structure. To extract factors that may distinguish the structures of ORs within the classes, we analyzed amino acid sequences of ORs within two classes in humans. Conserved residues from the analysis were classified into three different groups by conservation pattern in each class. We found that conservation profiles by structural regions in each class are different. To verify if these residues functionally distinguish the structures of ORs within two classes, two physicochemically differently conserved within the classes (PDCC) residues are experimentally tested by molecular dynamics simulations and expression on heterologous systems. The structure and the response of ORs by their agonist were changed significantly when the physicochemical feature in the residues was mutated by the one in the other class

Functionally associated classification of human olfactory receptor superfamily in the function associated manner

The odorant receptor (OR) is a main character in making olfactory sensory neurons (OSNs) activated by interacting with structurally diverse odorant molecules. Mammalian genomes of ORs are well established, and highly conserved regions within ORs have been identified. Numerous studies have focused on specific conserved amino acid motifs of ORs to examine their functional roles, which underlines their contribution to receptor functions. There are several variable regions among conserved motifs, and we found the patterns of variations within consensus amino acid sequences of the ORs. Based on our hypothesis that the related functions can be inferred from the pattern of variations in the conserved motifs of ORs, we performed hierarchical clustering of intact human OR proteins according to the concordance rate for selected conserved motifs. Human ORs were divided into two large groups, and classified into six sub-clusters according to the conserved patterns. We observed that the standard for dividing human ORs into two large groups is M (methionine) of PMLNPL/FIY, and verified the role of this region with selected representative OR models by conducting heterologous in vitro assay. This study proposed a new index for OR functions by presenting a conserved motif as a criterion for the classification of human OR families that cannot be fully explained by current classifications.1

Differential expression of Cul4a and Cul4b by NMDA-evoked neuronal activity

Cul4a and Cul4b are a core component of cullin-RING-based E3 ubiquitin ligase complex, which has multiple functions including DNA repair, chromatin remodeling and cell cycle regulation via ubiquitination of Ddb1-recruited target molecules. Although they have highly overlapped functions with high homology on their protein sequences as paralogue proteins, an irreplaceable role among them has been also proposed. Especially, Cul4b plays a critical role in neurodevelopment implicated with X-linked mental retardation unlike Cul4a. However, a specific role of CUL4A in nervous system has been veiled. In this study, we found that proteins of Cul4a and Cul4b are enriched in differential intracellular compartment of neurons. In addition, glutamate-evoked neuronal activity induces nuclear translocation of Cul4a with degradation of Cul4b. Pharmacological study shows that this process is mediated by intracellular calcium influx via NMDA receptor. Taken together, we suggest that Cul4a has an irreplaceable and differential role in nervous system compared to Cul4b.1

What is the function of ECL3, the shortest loop of the odorant receptor?

[No Abstract Available]FALS

The Third Extracellular Loop of Mammalian Odorant Receptors Is Involved in Ligand Binding

Mammals recognize chemicals in the air via G protein-coupled odorant receptors (ORs). In addition to their orthosteric binding site, other segments of these receptors modulate ligand recognition. Focusing on human hOR1A1, which is considered prototypical of class II ORs, we used a combination of molecular modeling, site-directed mutagenesis, and in vitro functional assays. We showed that the third extracellular loop of ORs (ECL3) contributes to ligand recognition and receptor activation. Indeed, site-directed mutations in ECL3 showed differential effects on the potency and efficacy of both carvones, citronellol, and 2-nonanone

Odorant Receptors Containing Conserved Amino Acid Sequences in Transmembrane Domain 7 Display Distinct Expression Patterns in Mammalian Tissues

Mammalian genomes are well established, and highly conserved regions within odorant receptors that are unique from other G-protein coupled receptors have been identified. Numerous functional studies have focused on specific conserved amino acids motifs; however, not all conserved motifs have been sufficiently characterized. Here, we identified a highly conserved 18 amino acid sequence motif within transmembrane domain seven (CAS-TM7) which was identified by aligning odorant receptor sequences. Next, we investigated the expression pattern and distribution of this conserved amino acid motif among a broad range of odorant receptors. To examine the localization of odorant receptor proteins, we used a sequence-specific peptide antibody against CAS-TM7 which is specific to odorant receptors across species. The specificity of this peptide antibody in recognizing odorant receptors has been confirmed in a heterologous in vitro system and a rat-based in vivo system. The CAS-TM7 odorant receptors localized with distinct patterns at each region of the olfactory epithelium; septum, endoturbinate and ectoturbinate. To our great interests, we found that the CAS-TM7 odorant receptors are primarily localized to the dorsal region of the olfactory bulb, coinciding with olfactory epithelium-based patterns. Also, these odorant receptors were ectopically expressed in the various non-olfactory tissues in an evolutionary constrained manner between human and rats. This study has characterized the expression patterns of odorant receptors containing particular amino acid motif in transmembrane domain 7, and which led to an intriguing possibility that the conserved motif of odorant receptors can play critical roles in other physiological functions as well as olfaction. © The Korean Society for Molecular and Cellular Biology. All rights reserved.FALS

Odorant receptors containing conserved amino acid sequences in transmembrane domain 7 display distinct expression patterns in mammalian tissues

[No Abstract Available]1