• Journal of the Korean Magnetic Resonance Society
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• v.23 no.3
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• pp.67-72
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• 2019

Caveolae are small plasma membrane invaginations that play many roles in signal transduction, endocytosis, mechanoprotection, lipid metabolism. The most important protein in caveolae is the integral membrane protein, caveolin, which is divided into three families such as caveolin 1, caveolin 2, and caveolin 3. Caveolin 1 and 3 are known to incorporate palmitate through linkage to three cysteine residues. Regulation of the protein palmitoylation cycle is important for the cellular processes such as intracellular localization of the target protein, membrane association, conformation, protein-protein interaction, and activity. However, the detailed aspect of individual palmitoylation has not been studied. In the present work, the role of each lipid modification at three cysteines was studied by NMR. Our results suggest that each lipid modification at the natively palmitoylation site has its own roles. For example, lipidations to C106 and C129 are play a role in structural stabilization, however, interestingly, lipid modification to C116 interrupts the structural stabilization.

• Analytical Science and Technology
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• v.29 no.1
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• pp.10-18
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• 2016

Phosphodiesterase (PDE) plays an important role in cAMP-mediated signaling within cells. We previously showed that the long-form of Aplysia PDE4 (ApPDE4) was localized in the plasma membrane and the presynaptic terminal in Aplysia sensory neurons, and the 16 N-terminal amino acid was sufficient for this targeting process. In this study, we characterized the cellular localization of various ApPDE4 mutants. We first identified the roles of each amino acid within the group of 16 N-terminal amino acids of long-form ApPDE4. As a result, we were able to identify various mutants that were localized to both the plasma membrane and the Golgi complex, Golgi only, or both the endoplasmic reticulum (ER) and the Golgi complex. To examine the role of palmitoylation on the cellular localization of ApPDE4 mutants, 2-bromo palmitate (2-BR) was used as a treatment. As a result, in the presence of 2-BR, the plasma membrane targeting of many mutants was impaired, indicating that palmitoylation was involved in the plasma membrane targeting of the mutants. We also found that PI4P play crucial roles in the Golgi targeting of (N16,C3S/VV/G)-mRFP, L(N16,C3S/LFS/R)-mRFP, and L(N16,EPL/R)-mRFP.

• BMB Reports
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• v.42 no.1
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• pp.1-5
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• 2009

Synaptic vesicles (SVs) are key structures for synaptic transmission in neurons. Numerous membrane-associated proteins are sorted from the Golgi complex to the axon and the presynaptic terminal. Protein-protein and protein-lipid interactions are involved with SV targeting in neurons. Interestingly, many SV proteins have lipid binding capability, primarily with either cholesterol or phosphoinositides (PIs). As examples, the major SV protein synaptophysin can bind to cholesterol, a major lipid component in SVs, while several other SV proteins, including synaptotagmin, can bind to PIs. Thus, lipid-protein binding plays a key role for the SV targeting of synaptic proteins. In addition, numerous SV proteins can be palmitoylated. Palmitoylation is thought to be another synaptic targeting signal. Here, we briefly describe the relationship between lipid binding and SV targeting.

• Biomolecules & Therapeutics
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• v.25 no.1
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• pp.26-43
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• 2017

Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with ${\beta}$-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis.

• Biomedical Science Letters
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• v.28 no.2
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• pp.59-66
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• 2022

The Cyr61/CTGF/NOV (CCN) family is dynamically expressed in various tissues, including the nervous system, from the prenatal period to adulthood. However, major studies have been conducted only in limited fields, such as the cardiovascular and muscular systems, skeletal development, and cancer. In addition, although the CCN family is a secretory protein, very few studies have described its mechanism of secretion. Recently, it has been suggested that overexpression of CCN3 or intracellular accumulation due to problems in the secretory pathway can inhibit neuronal axonal growth. In this review, we have briefly summarized the structure and characteristics of the CCN family and its related diseases, with particular emphasis on the secretory mechanism and modifiers of the CCN family, newly identified in the nervous system.

• Biomedical Science Letters
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• v.24 no.2
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• pp.64-75
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• 2018

CCN3 (also known as NOV, Nephroblastoma overexpressed) proteins are involved in various pathologies during different developmental stages. We have previously shown that intracellular levels and normal extracellular secretion of CCN3 are important for neuronal differentiation. Furthermore, we demonstrated that a single amino acid in the CCN3 TSP-1 domain is important for extracellular secretion and that palmitoylation of CCN3 is required in this process. However, the effect of abnormal CCN3 accumulation on cells remains to be studied. Here, we found mutations in the TSP-1 domain of CCN3 that led to intracellular accumulation and abnormal aggregation of CCN3. It was observed that this mutation resulted in a phenomenon similar to neurodegeneration when overexpressed in the developing mouse cortex. This mutation also confirmed the activation of apoptotic gene expression in Neuro2a cells. In addition, we confirmed the in vivo transcriptional changes induced by this mutation using microarray analysis. We observed a significant increase in the expression of Anp32a, an apoptosis-related gene. Collectively, these results indicate that a single mutation in CCN3 can lead to abnormal cell death if it shows intracellular accumulation and abnormal aggregation.

• Fisheries and Aquatic Sciences
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• v.12 no.4
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• pp.265-275
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• 2009

G Protein-coupled receptors (GPCRs) mediating wide ranges of physiological responses is one of the most attractive targets for drug development. Rhodopsin, a dim-light photoreceptor, has been extensively used as a model system for structural and functional study of GPCRs. Fish have rhodopsin finely-tuned to their habitats where the intensity and the wavelength of lights are changed depending on its water-depth. To study the detailed molecular characteristics of GPCR architecture and to understand the fishery light-sensing system, genes encoding rod opsins were isolated from fishes living under different photic environments. Full-length rod opsin genes were obtained by combination of PCR amplification and DNA walking strategy of genomic DNA isolated from olive flounder, P. olivaceus, Japanese eel, A. japonica, and Common carp C. carpio. Deduced amino acid sequences showed a typical feature of rod opsins including the sites for Schiffs base formation (Lys296) and its counter ion (Glu113), disulfide formation (Cys110 and Cys187), and palmitoylation (Cys322 and Cys323) although Cys322 is replaced by Phe in Japanese eel. Comparison of opsins by amino acid sequence alignment indicated the closest similarity between P. olivaceus and H. hippoglossus (94%), A. japonica and A. anguilla (98%), and C. carpio and C. auratus (95%), respectively.

• Fisheries and Aquatic Sciences
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• v.10 no.1
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• pp.8-15
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• 2007

Rhodopsin, a dim-light receptor, is a model system for the study of G protein-coupled receptors that transduce extracellular signals into cells. To study the molecular mechanisms of visual systems in fish, the rod opsin gene of olive flounder Paralichthys olivaceus was characterized. The full-length P. olivaceus opsin gene was obtained by PCR amplification of genomic DNA, as well as cDNA synthesis. A comparison of clones obtained from both methods indicated that the olive flounder rod opsin gene lacks introns. Sequence analysis of the opsin gene indicated that it contains a 1,056-bp open reading frame encoding 352 amino acids. The deduced amino acid sequence contains features of typical rod opsins, such as sites for Schiff's base formation (K296) and its counterion (E113), disulfide formation (C110 and C187), and palmitoylation (C322 and C323). An opsin sequence alignment showed the highest similarity between P. olivaceus and Solea solea (95.1%), followed by Hippoglossus hippoglossus (94.5%). An opsin phylogenetic tree revealed a close relationship between olive flounder and teleost rod opsins.