Pub Date : 2023-01-25eCollection Date: 2023-01-01DOI: 10.2142/biophysico.bppb-v20.0005
Tomoyoshi Seto
The GABAA receptor (GABAAR) is a target channel for the loss of awareness of general anesthesia. General anesthetic (GA) spans a wide range of chemical structures, such as monatomic molecules, barbital acids, phenols, ethers, and alkanes. GA has a weak binding affinity, and the affinity has a characteristic that correlates with the solubility in olive oil rather than the molecular shape. The GA binding site of GABAAR is common to GAs and exists in the transmembrane domain of the GABAAR intersubunit. In this study, the mechanism of GA binding, which allows binding of various GAs with intersubunit selectivity, was elucidated from the hydration analysis of the binding site. Regardless of the diverse GA chemical structures, a strong correlation was observed between the binding free energy and total dehydration number of the binding process. The GA binding free energy was more involved in the binding dehydration and showed molecular recognition that allowed for the binding of various GA structures via binding site hydration. We regarded the GA substitution for the interfacial water molecule of the binding site as a dissolution into the interfacial hydration layer. The elucidation of the GA binding mechanism mediated by hydration at the GABAAR common binding site provides a rationale for the combined use of anesthetics in medical practice and its combination adjustments via drug interactions.
GABAA 受体(GABAAR)是全身麻醉失去知觉的目标通道。全身麻醉剂(GA)的化学结构多种多样,如单原子分子、巴比妥酸、酚类、醚类和烷类。GA 具有较弱的结合亲和力,这种亲和力的特征与在橄榄油中的溶解度而不是分子形状有关。GABAAR 的 GA 结合位点是 GAs 的共性,存在于 GABAAR 亚基间的跨膜结构域中。本研究通过对该结合位点的水合分析,阐明了 GA 的结合机制,该机制使各种 GA 的结合具有亚基间选择性。无论 GA 的化学结构如何变化,在结合过程中,结合自由能与总脱水数之间都存在很强的相关性。GA 的结合自由能更多地参与了结合脱水过程,并显示出分子识别能力,通过结合位点的水合作用使各种 GA 结构得以结合。我们认为,GA 对结合位点界面水分子的取代是对界面水合层的溶解。通过阐明 GABAAR 共同结合位点水合介导的 GA 结合机制,为医疗实践中麻醉剂的联合使用及其通过药物相互作用进行联合调整提供了理论依据。
{"title":"General anesthetic binding mode via hydration with weak affinity and molecular discrimination: General anesthetic dissolution in interfacial water of the common binding site of GABA<sub>A</sub> receptor.","authors":"Tomoyoshi Seto","doi":"10.2142/biophysico.bppb-v20.0005","DOIUrl":"10.2142/biophysico.bppb-v20.0005","url":null,"abstract":"<p><p>The GABA<sub>A</sub> receptor (GABA<sub>A</sub>R) is a target channel for the loss of awareness of general anesthesia. General anesthetic (GA) spans a wide range of chemical structures, such as monatomic molecules, barbital acids, phenols, ethers, and alkanes. GA has a weak binding affinity, and the affinity has a characteristic that correlates with the solubility in olive oil rather than the molecular shape. The GA binding site of GABA<sub>A</sub>R is common to GAs and exists in the transmembrane domain of the GABA<sub>A</sub>R intersubunit. In this study, the mechanism of GA binding, which allows binding of various GAs with intersubunit selectivity, was elucidated from the hydration analysis of the binding site. Regardless of the diverse GA chemical structures, a strong correlation was observed between the binding free energy and total dehydration number of the binding process. The GA binding free energy was more involved in the binding dehydration and showed molecular recognition that allowed for the binding of various GA structures via binding site hydration. We regarded the GA substitution for the interfacial water molecule of the binding site as a dissolution into the interfacial hydration layer. The elucidation of the GA binding mechanism mediated by hydration at the GABA<sub>A</sub>R common binding site provides a rationale for the combined use of anesthetics in medical practice and its combination adjustments via drug interactions.</p>","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10941959/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74244184","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-25eCollection Date: 2023-03-21DOI: 10.2142/biophysico.bppb-v20.s009
Kazumi Shimono, Norbert A Dencher
{"title":"Recent advances in signaling and activation mechanism in microbial rhodopsins: Report for the session 6 at the 19<sup>th</sup> International Conference on Retinal Proteins.","authors":"Kazumi Shimono, Norbert A Dencher","doi":"10.2142/biophysico.bppb-v20.s009","DOIUrl":"10.2142/biophysico.bppb-v20.s009","url":null,"abstract":"","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10865883/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85327552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rhodopsins have been extensively employed for optogenetic regulation of bioelectrical activity of excitable cells and other cellular processes across biological systems. Various strategies have been adopted to attune the cellular processes at the desired subcellular compartment (plasma membrane, endoplasmic reticulum, Golgi, mitochondria, lysosome) within the cell. These strategies include-adding signal sequences, tethering peptides, specific interaction sites, or mRNA elements at different sites in the optogenetic proteins for plasma membrane integration and subcellular targeting. However, a single approach for organelle optogenetics was not suitable for the relevant optogenetic proteins and often led to the poor expression, mislocalization, or altered physical and functional properties. Therefore, the current study is focused on the native subcellular targeting machinery of algal rhodopsins. The N- and C-terminus signal prediction led to the identification of rhodopsins with diverse organelle targeting signal sequences for the nucleus, mitochondria, lysosome, endosome, vacuole, and cilia. Several identified channelrhodopsins and ion-pumping rhodopsins possess effector domains associated with DNA metabolism (repair, replication, and recombination) and gene regulation. The identified algal rhodopsins with diverse effector domains and encoded native subcellular targeting sequences hold immense potential to establish expanded organelle optogenetic regulation and associated cellular signaling.
在整个生物系统中,人们已广泛利用罗得蛋白对可兴奋细胞的生物电活动和其他细胞过程进行光遗传调控。人们采用了各种策略来调整细胞内所需亚细胞区室(质膜、内质网、高尔基体、线粒体、溶酶体)的细胞过程。这些策略包括在光遗传蛋白的不同部位添加信号序列、系留肽、特定的相互作用位点或 mRNA 元件,以实现质膜整合和亚细胞靶向。然而,单一的细胞器光遗传学方法并不适合相关的光遗传蛋白,往往会导致表达不良、定位错误或物理和功能特性改变。因此,目前的研究侧重于藻类视蛋白的原生亚细胞靶向机制。通过对 N 端和 C 端信号的预测,发现了具有多种细胞器靶向信号序列的藻类视蛋白,包括细胞核、线粒体、溶酶体、内质体、液泡和纤毛。已发现的几种通道视蛋白和离子泵视蛋白具有与 DNA 代谢(修复、复制和重组)和基因调控有关的效应结构域。已发现的藻类视网膜蛋白具有不同的效应结构域和编码的本地亚细胞靶向序列,在建立扩展的细胞器光遗传调控和相关细胞信号传导方面具有巨大潜力。
{"title":"Algal rhodopsins encoding diverse signal sequence holds potential for expansion of organelle optogenetics.","authors":"Kumari Sushmita, Sunita Sharma, Manish Singh Kaushik, Suneel Kateriya","doi":"10.2142/biophysico.bppb-v20.s008","DOIUrl":"10.2142/biophysico.bppb-v20.s008","url":null,"abstract":"<p><p>Rhodopsins have been extensively employed for optogenetic regulation of bioelectrical activity of excitable cells and other cellular processes across biological systems. Various strategies have been adopted to attune the cellular processes at the desired subcellular compartment (plasma membrane, endoplasmic reticulum, Golgi, mitochondria, lysosome) within the cell. These strategies include-adding signal sequences, tethering peptides, specific interaction sites, or mRNA elements at different sites in the optogenetic proteins for plasma membrane integration and subcellular targeting. However, a single approach for organelle optogenetics was not suitable for the relevant optogenetic proteins and often led to the poor expression, mislocalization, or altered physical and functional properties. Therefore, the current study is focused on the native subcellular targeting machinery of algal rhodopsins. The N- and C-terminus signal prediction led to the identification of rhodopsins with diverse organelle targeting signal sequences for the nucleus, mitochondria, lysosome, endosome, vacuole, and cilia. Several identified channelrhodopsins and ion-pumping rhodopsins possess effector domains associated with DNA metabolism (repair, replication, and recombination) and gene regulation. The identified algal rhodopsins with diverse effector domains and encoded native subcellular targeting sequences hold immense potential to establish expanded organelle optogenetic regulation and associated cellular signaling.</p>","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10865886/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73580613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-24eCollection Date: 2023-03-21DOI: 10.2142/biophysico.bppb-v20.s007
Jonathan R Church, Jógvan Magnus Haugaard Olsen, Igor Schapiro
Multiscale simulations have been established as a powerful tool to calculate and predict excitation energies in complex systems such as photoreceptor proteins. In these simulations the chromophore is typically treated using quantum mechanical (QM) methods while the protein and surrounding environment are described by a classical molecular mechanics (MM) force field. The electrostatic interactions between these regions are often treated using electrostatic embedding where the point charges in the MM region polarize the QM region. A more sophisticated treatment accounts also for the polarization of the MM region. In this work, the effect of such a polarizable embedding on excitation energies was benchmarked and compared to electrostatic embedding. This was done for two different proteins, the lipid membrane-embedded jumping spider rhodopsin and the soluble cyanobacteriochrome Slr1393g3. It was found that the polarizable embedding scheme produces absorption maxima closer to experimental values. The polarizable embedding scheme was also benchmarked against expanded QM regions and found to be in qualitative agreement. Treating individual residues as polarizable recovered between 50% and 71% of the QM improvement in the excitation energies, depending on the system. A detailed analysis of each amino acid residue in the chromophore binding pocket revealed that aromatic residues result in the largest change in excitation energy compared to the electrostatic embedding. Furthermore, the computational efficiency of polarizable embedding allowed it to go beyond the binding pocket and describe a larger portion of the environment, further improving the results.
{"title":"Induction effects on the absorption maxima of photoreceptor proteins.","authors":"Jonathan R Church, Jógvan Magnus Haugaard Olsen, Igor Schapiro","doi":"10.2142/biophysico.bppb-v20.s007","DOIUrl":"10.2142/biophysico.bppb-v20.s007","url":null,"abstract":"<p><p>Multiscale simulations have been established as a powerful tool to calculate and predict excitation energies in complex systems such as photoreceptor proteins. In these simulations the chromophore is typically treated using quantum mechanical (QM) methods while the protein and surrounding environment are described by a classical molecular mechanics (MM) force field. The electrostatic interactions between these regions are often treated using electrostatic embedding where the point charges in the MM region polarize the QM region. A more sophisticated treatment accounts also for the polarization of the MM region. In this work, the effect of such a polarizable embedding on excitation energies was benchmarked and compared to electrostatic embedding. This was done for two different proteins, the lipid membrane-embedded jumping spider rhodopsin and the soluble cyanobacteriochrome Slr1393g3. It was found that the polarizable embedding scheme produces absorption maxima closer to experimental values. The polarizable embedding scheme was also benchmarked against expanded QM regions and found to be in qualitative agreement. Treating individual residues as polarizable recovered between 50% and 71% of the QM improvement in the excitation energies, depending on the system. A detailed analysis of each amino acid residue in the chromophore binding pocket revealed that aromatic residues result in the largest change in excitation energy compared to the electrostatic embedding. Furthermore, the computational efficiency of polarizable embedding allowed it to go beyond the binding pocket and describe a larger portion of the environment, further improving the results.</p>","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10865876/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78251426","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-19eCollection Date: 2023-03-21DOI: 10.2142/biophysico.bppb-v20.s006
Mikio Kataoka
It marked half a century since the discovery of bacteriorhodopsin two years ago. On this occasion, I have revisited historically important diffraction studies of this membrane protein, based on my recollections. X-ray diffraction and electron diffraction, and electron microscopy, described the low-resolution structure of bacteriorhodopsin within the purple membrane. Neutron diffraction was effective to assign the helical regions in the primary structure with 7 rods revealed by low-resolution structure as well as to describe the retinal position. Substantial conformational changes upon light illumination were clarified by the structures of various photointermediates. Early trials of time-resolved studies were also introduced. Models for the mechanism of light-driven proton pump based on the low-resolution structural studies are also described. Significantly, they are not far from the today's understanding. I believe that the spirit of the early research scientists in this field and the essence of their studies, which constitute the foundations of the field, still actively fertilizes current membrane protein research.
{"title":"Structural studies of bacteriorhodopsin in BC era.","authors":"Mikio Kataoka","doi":"10.2142/biophysico.bppb-v20.s006","DOIUrl":"10.2142/biophysico.bppb-v20.s006","url":null,"abstract":"<p><p>It marked half a century since the discovery of bacteriorhodopsin two years ago. On this occasion, I have revisited historically important diffraction studies of this membrane protein, based on my recollections. X-ray diffraction and electron diffraction, and electron microscopy, described the low-resolution structure of bacteriorhodopsin within the purple membrane. Neutron diffraction was effective to assign the helical regions in the primary structure with 7 rods revealed by low-resolution structure as well as to describe the retinal position. Substantial conformational changes upon light illumination were clarified by the structures of various photointermediates. Early trials of time-resolved studies were also introduced. Models for the mechanism of light-driven proton pump based on the low-resolution structural studies are also described. Significantly, they are not far from the today's understanding. I believe that the spirit of the early research scientists in this field and the essence of their studies, which constitute the foundations of the field, still actively fertilizes current membrane protein research.</p>","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10865857/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77509416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-11eCollection Date: 2023-03-21DOI: 10.2142/biophysico.bppb-v20.s005
Yuji Furutani, Chii-Shen Yang
{"title":"Ion-transporting mechanism in microbial rhodopsins: Mini-review relating to the session 5 at the 19th International Conference on Retinal Proteins.","authors":"Yuji Furutani, Chii-Shen Yang","doi":"10.2142/biophysico.bppb-v20.s005","DOIUrl":"10.2142/biophysico.bppb-v20.s005","url":null,"abstract":"","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10865854/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90680326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Recent small-angle X-ray scattering (SAXS) for biological macromolecules (BioSAXS) is generally combined with size-exclusion chromatography (SEC-SAXS) at synchrotron facilities worldwide. For SEC-SAXS analysis, the final scattering profile for the target molecule is calculated from a large volume of continuously collected data. It would be ideal to automate this process; however, several complex problems exist regarding data measurement and analysis that have prevented automation. Here, we developed the analytical software MOLASS (Matrix Optimization with Low-rank factorization for Automated analysis of SEC-SAXS) to automatically calculate the final scattering profiles for solution structure analysis of target molecules. In this paper, the strategies for automatic analysis of SEC-SAXS data are described, including correction of baseline-drift using a low percentile method, optimization of peak decompositions composed of multiple scattering components using modified Gaussian fitting against the chromatogram, and rank determination for extrapolation to infinite dilution. In order to easily calculate each scattering component, the Moore-Penrose pseudo-inverse matrix is adopted as a basic calculation. Furthermore, this analysis method, in combination with UV-visible spectroscopy, led to better results in terms of accuracy in peak decomposition. Therefore, MOLASS will be able to smoothly suggest to users an accurate scattering profile for the subsequent structural analysis.
目前,生物大分子的小角度x射线散射(SAXS)技术在世界范围内的同步加速器上普遍采用与粒径隔离色谱(SEC-SAXS)相结合的方法。对于SEC-SAXS分析,从大量连续收集的数据中计算目标分子的最终散射曲线。将这一过程自动化是理想的;然而,在数据测量和分析方面存在一些复杂的问题,这些问题阻碍了自动化。在此,我们开发了分析软件MOLASS (Matrix Optimization with Low-rank factorization for Automated analysis of SEC-SAXS),用于自动计算目标分子溶液结构分析的最终散射曲线。本文介绍了SEC-SAXS数据自动分析的策略,包括使用低百分位法校正基线漂移,使用改进的高斯拟合针对色谱图优化由多个散射分量组成的峰分解,以及用于无限稀释外推的等级确定。为了方便计算各个散射分量,采用Moore-Penrose伪逆矩阵作为基本计算。此外,该分析方法与紫外-可见光谱相结合,在峰分解的准确性方面取得了更好的结果。因此,MOLASS将能够顺利地向用户提供准确的散射剖面,用于后续的结构分析。
{"title":"MOLASS: Software for automatic processing of matrix data obtained from small-angle X-ray scattering and UV-visible spectroscopy combined with size-exclusion chromatography.","authors":"Kento Yonezawa, Masatsuyo Takahashi, Keiko Yatabe, Yasuko Nagatani, Nobutaka Shimizu","doi":"10.2142/biophysico.bppb-v20.0001","DOIUrl":"https://doi.org/10.2142/biophysico.bppb-v20.0001","url":null,"abstract":"<p><p>Recent small-angle X-ray scattering (SAXS) for biological macromolecules (BioSAXS) is generally combined with size-exclusion chromatography (SEC-SAXS) at synchrotron facilities worldwide. For SEC-SAXS analysis, the final scattering profile for the target molecule is calculated from a large volume of continuously collected data. It would be ideal to automate this process; however, several complex problems exist regarding data measurement and analysis that have prevented automation. Here, we developed the analytical software MOLASS (Matrix Optimization with Low-rank factorization for Automated analysis of SEC-SAXS) to automatically calculate the final scattering profiles for solution structure analysis of target molecules. In this paper, the strategies for automatic analysis of SEC-SAXS data are described, including correction of baseline-drift using a low percentile method, optimization of peak decompositions composed of multiple scattering components using modified Gaussian fitting against the chromatogram, and rank determination for extrapolation to infinite dilution. In order to easily calculate each scattering component, the Moore-Penrose pseudo-inverse matrix is adopted as a basic calculation. Furthermore, this analysis method, in combination with UV-visible spectroscopy, led to better results in terms of accuracy in peak decomposition. Therefore, MOLASS will be able to smoothly suggest to users an accurate scattering profile for the subsequent structural analysis.</p>","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/6c/80/20_e200001.PMC10203098.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9518816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.2142/biophysico.bppb-v20.0003
Kanta Fujimoto, Kosuke Inada, Kotaro Oka, Etsuro Ito
Some evidence suggests that oxytocin, which is a neuropeptide conventionally thought to be synthesized in the hypothalamus and released by the posterior pituitary, is generated in peripheral keratinocytes, but the details are lacking and the mRNA analysis is further required. Oxytocin and neurophysin I are generated together as cleavage products after splitting the precursor molecule, preprooxyphysin. To confirm that oxytocin and neurophysin I are also generated in the peripheral keratinocytes, it must first be clarified that these molecules contained in peripheral keratinocytes did not originate in the posterior pituitary gland and then the expression of oxytocin and neurophysin I mRNAs must be established in keratinocytes. Therefore, we attempted to quantify preprooxyphysin mRNA in keratinocytes using various primers. Using real-time PCR, we observed that the mRNAs of both oxytocin and neurophysin I were located in keratinocytes. However, the mRNA amounts of oxytocin, neurophysin I, and preprooxyphysin were too small to confirm their co-existence in keratinocytes. Thus, we had to further determine whether the PCR-amplified sequence was identical to preprooxyphysin. The PCR products analyzed by DNA sequencing were identical to preprooxyphysin, finally determining the co-existence of both oxytocin and neurophysin I mRNAs in keratinocytes. In addition, the immunocytochemical experiments showed that oxytocin and neurophysin I proteins were located in keratinocytes. These results of the present study provided further support indicating that oxytocin and neurophysin I are generated in peripheral keratinocytes.
一些证据表明,催产素是一种通常被认为在下丘脑合成并由垂体后叶释放的神经肽,它是在周围角质形成细胞中产生的,但缺乏细节,需要进一步的mRNA分析。催产素和神经physin I在前体分子pre - prooxyphysin分裂后作为裂解产物一起产生。为了证实催产素和神经physin I也在外周角质形成细胞中产生,首先必须澄清外周角质形成细胞中包含的这些分子并非起源于垂体后腺,然后必须在角质形成细胞中建立催产素和神经physin I mrna的表达。因此,我们尝试使用不同的引物定量角化细胞中的前氧合素mRNA。通过实时PCR,我们观察到催产素和神经physin I的mrna都位于角化细胞中。然而,在角化细胞中,催产素、神经physin I和preprooxyphysin的mRNA量太少,无法证实它们的共存。因此,我们必须进一步确定pcr扩增的序列是否与preprooxyphysin相同。通过DNA测序分析的PCR产物与前prooxyphysin相同,最终确定了角化细胞中催产素和神经physin I mrna共存。此外,免疫细胞化学实验表明,催产素和神经physin I蛋白位于角化细胞中。本研究的这些结果进一步支持了催产素和神经physin I是在周围角质形成细胞中产生的。
{"title":"Revisiting oxytocin generation in keratinocytes.","authors":"Kanta Fujimoto, Kosuke Inada, Kotaro Oka, Etsuro Ito","doi":"10.2142/biophysico.bppb-v20.0003","DOIUrl":"https://doi.org/10.2142/biophysico.bppb-v20.0003","url":null,"abstract":"<p><p>Some evidence suggests that oxytocin, which is a neuropeptide conventionally thought to be synthesized in the hypothalamus and released by the posterior pituitary, is generated in peripheral keratinocytes, but the details are lacking and the mRNA analysis is further required. Oxytocin and neurophysin I are generated together as cleavage products after splitting the precursor molecule, preprooxyphysin. To confirm that oxytocin and neurophysin I are also generated in the peripheral keratinocytes, it must first be clarified that these molecules contained in peripheral keratinocytes did not originate in the posterior pituitary gland and then the expression of oxytocin and neurophysin I mRNAs must be established in keratinocytes. Therefore, we attempted to quantify preprooxyphysin mRNA in keratinocytes using various primers. Using real-time PCR, we observed that the mRNAs of both oxytocin and neurophysin I were located in keratinocytes. However, the mRNA amounts of oxytocin, neurophysin I, and preprooxyphysin were too small to confirm their co-existence in keratinocytes. Thus, we had to further determine whether the PCR-amplified sequence was identical to preprooxyphysin. The PCR products analyzed by DNA sequencing were identical to preprooxyphysin, finally determining the co-existence of both oxytocin and neurophysin I mRNAs in keratinocytes. In addition, the immunocytochemical experiments showed that oxytocin and neurophysin I proteins were located in keratinocytes. These results of the present study provided further support indicating that oxytocin and neurophysin I are generated in peripheral keratinocytes.</p>","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d3/c5/20_e200003.PMC10205573.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9531814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.2142/biophysico.bppb-v20.0037
Tomohiro Shima, Kumiko Hayashi
{"title":"The third Japan-U.S. symposium on motor proteins and associated single-molecule biophysics","authors":"Tomohiro Shima, Kumiko Hayashi","doi":"10.2142/biophysico.bppb-v20.0037","DOIUrl":"https://doi.org/10.2142/biophysico.bppb-v20.0037","url":null,"abstract":"","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135911480","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ciliary bending movements are powered by motor protein axonemal dyneins. They are largely classified into two groups, inner-arm dynein and outer-arm dynein. Outer-arm dynein, which is important for the elevation of ciliary beat frequency, has three heavy chains (α, β, and γ), two intermediate chains, and more than 10 light chains in green algae, Chlamydomonas. Most of intermediate chains and light chains bind to the tail regions of heavy chains. In contrast, the light chain LC1 was found to bind to the ATP-dependent microtubule-binding domain of outer-arm dynein γ-heavy chain. Interestingly, LC1 was also found to interact with microtubules directly, but it reduces the affinity of the microtubule-binding domain of γ-heavy chain for microtubules, suggesting the possibility that LC1 may control ciliary movement by regulating the affinity of outer-arm dyneins for microtubules. This hypothesis is supported by the LC1 mutant studies in Chlamydomonas and Planaria showing that ciliary movements in LC1 mutants were disordered with low coordination of beating and low beat frequency. To understand the molecular mechanism of the regulation of outer-arm dynein motor activity by LC1, X-ray crystallography and cryo-electron microscopy have been used to determine the structure of the light chain bound to the microtubule-binding domain of γ-heavy chain. In this review article, we show the recent progress of structural studies of LC1, and suggest the regulatory role of LC1 in the motor activity of outer-arm dyneins. This review article is an extended version of the Japanese article, The Complex of Outer-arm Dynein Light Chain-1 and the Microtubule-binding Domain of the Heavy Chain Shows How Axonemal Dynein Tunes Ciliary Beating, published in SEIBUTSU BUTSURI Vol. 61, p. 20-22 (2021).
纤毛弯曲运动是由运动蛋白轴突动力驱动的。它们大致分为两类,臂内动力蛋白和臂外动力蛋白。在绿藻、衣藻中,外臂动力蛋白具有3条重链(α、β和γ)、2条中间链和10多条轻链,对纤毛跳动频率的升高起重要作用。大多数中间链和轻链结合在重链的尾部。相比之下,轻链LC1被发现与外臂动力蛋白γ-重链的atp依赖的微管结合域结合。有趣的是,LC1也被发现与微管直接相互作用,但它降低了γ-重链微管结合域对微管的亲和力,这表明LC1可能通过调节外臂动力蛋白对微管的亲和力来控制纤毛运动。这一假设得到了衣藻和涡虫LC1突变体研究的支持,表明LC1突变体的纤毛运动紊乱,搏动协调性低,搏动频率低。为了解LC1调控外臂动力蛋白运动活性的分子机制,利用x射线晶体学和低温电镜对γ-重链微管结合域的轻链结构进行了测定。在这篇综述文章中,我们展示了LC1的结构研究的最新进展,并提出了LC1在外臂动力蛋白运动活动中的调节作用。这篇综述文章是日本文章《外臂动力蛋白轻链-1复合物和重链微管结合域显示轴突动力蛋白如何调节纤毛跳动》的扩展版,发表于SEIBUTSU BUTSURI Vol. 61, p. 20-22(2021)。
{"title":"Regulation of motor activity of ciliary outer-arm dynein by the light chain 1; Implications from the structure of the light chain bound to the microtubule-binding domain of the heavy chain.","authors":"Toshiki Yagi, Akiyuki Toda, Muneyoshi Ichikawa, Genji Kurisu","doi":"10.2142/biophysico.bppb-v20.0008","DOIUrl":"https://doi.org/10.2142/biophysico.bppb-v20.0008","url":null,"abstract":"<p><p>Ciliary bending movements are powered by motor protein axonemal dyneins. They are largely classified into two groups, inner-arm dynein and outer-arm dynein. Outer-arm dynein, which is important for the elevation of ciliary beat frequency, has three heavy chains (α, β, and γ), two intermediate chains, and more than 10 light chains in green algae, <i>Chlamydomonas</i>. Most of intermediate chains and light chains bind to the tail regions of heavy chains. In contrast, the light chain LC1 was found to bind to the ATP-dependent microtubule-binding domain of outer-arm dynein γ-heavy chain. Interestingly, LC1 was also found to interact with microtubules directly, but it reduces the affinity of the microtubule-binding domain of γ-heavy chain for microtubules, suggesting the possibility that LC1 may control ciliary movement by regulating the affinity of outer-arm dyneins for microtubules. This hypothesis is supported by the LC1 mutant studies in <i>Chlamydomonas</i> and Planaria showing that ciliary movements in LC1 mutants were disordered with low coordination of beating and low beat frequency. To understand the molecular mechanism of the regulation of outer-arm dynein motor activity by LC1, X-ray crystallography and cryo-electron microscopy have been used to determine the structure of the light chain bound to the microtubule-binding domain of γ-heavy chain. In this review article, we show the recent progress of structural studies of LC1, and suggest the regulatory role of LC1 in the motor activity of outer-arm dyneins. This review article is an extended version of the Japanese article, The Complex of Outer-arm Dynein Light Chain-1 and the Microtubule-binding Domain of the Heavy Chain Shows How Axonemal Dynein Tunes Ciliary Beating, published in SEIBUTSU BUTSURI Vol. 61, p. 20-22 (2021).</p>","PeriodicalId":8976,"journal":{"name":"Biophysics and Physicobiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/94/e3/20_e200008.PMC10205576.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9526629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}