Pub Date : 2024-10-09DOI: 10.1016/j.jsb.2024.108135
Olivier Duverger, Janice S. Lee
Tooth enamel is a fascinating tissue with exceptional biomechanical properties that allow it to last for a lifetime. In this mini review, we discuss the unique embryonic origin of this highly mineralized tissue, the complex differentiation process that leads to its “construction” (amelogenesis), and the various genetic conditions that lead to impaired enamel development in humans (amelogenesis imperfecta). Tremendous progress was made in the last 30 years in understanding the molecular and cellular mechanism that leads to normal and pathologic enamel development. However, several aspects of amelogenesis remain to be elucidated and the function of many genes associated with amelogenesis imperfecta still needs to be decoded.
{"title":"The intricacies of tooth enamel: Embryonic origin, development and human genetics","authors":"Olivier Duverger, Janice S. Lee","doi":"10.1016/j.jsb.2024.108135","DOIUrl":"10.1016/j.jsb.2024.108135","url":null,"abstract":"<div><div>Tooth enamel is a fascinating tissue with exceptional biomechanical properties that allow it to last for a lifetime. In this mini review, we discuss the unique embryonic origin of this highly mineralized tissue, the complex differentiation process that leads to its “construction” (amelogenesis), and the various genetic conditions that lead to impaired enamel development in humans (amelogenesis imperfecta). Tremendous progress was made in the last 30 years in understanding the molecular and cellular mechanism that leads to normal and pathologic enamel development. However, several aspects of amelogenesis remain to be elucidated and the function of many genes associated with amelogenesis imperfecta still needs to be decoded.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108135"},"PeriodicalIF":3.0,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.jsb.2024.108130
Maciej Nielipinski, Dominika Nielipinska, Agnieszka J. Pietrzyk-Brzezinska, Bartosz Sekula
Arginine is an important amino acid in plants, as it not only plays a structural role and serves as nitrogen storage but is also a precursor for various molecules, including polyamines and proline. Arginine is produced by argininosuccinate lyase (ASL) which catalyzes the cleavage of argininosuccinate to arginine and fumarate. ASL belongs to the fumarate lyase family and while many members of this family were well-characterized, little is known about plant ASLs. Here we present the first crystal structures of ASL from the model plant, Arabidopsis thaliana (AtASL). One of the structures represents the unliganded form of the AtASL homotetramer. The other structure, obtained from a crystal soaked in argininosuccinate, accommodates the substrate or the reaction products in one of four active sites of the AtASL tetramer. Each active site is located at the interface of three neighboring protomers. The AtASL structure with ligands allowed us to analyze the enzyme-substrate and the enzyme-product interactions in detail. Furthermore, based on our analyses, we describe residues of AtASL crucial for catalysis. The structure of AtASL gives the rationale for the open-to-close transition of the GSS mobile loop and indicates the importance of serine 333 from this loop for the enzymatic action of the enzyme. Finally, we supplemented the structural data with the identification of sequence motifs characteristic for ASLs.
精氨酸是植物体内的一种重要氨基酸，因为它不仅起着结构作用和储氮作用，还是多胺和脯氨酸等多种分子的前体。精氨酸由精琥珀酸裂解酶（ASL）生成，该酶催化精琥珀酸裂解为精氨酸和富马酸。ASL 属于富马酸裂解酶家族，尽管该家族中的许多成员都已被很好地描述，但人们对植物 ASL 却知之甚少。在这里，我们首次展示了来自模式植物拟南芥（Arabidopsis thaliana，AtASL）的 ASL 晶体结构。其中一个结构代表了 AtASL 同源四聚体的非连接形式。另一个结构是从浸泡在精琥珀酸中的晶体中获得的，它将底物或反应产物容纳在 AtASL 四聚体的四个活性位点之一。每个活性位点都位于三个相邻原体的界面上。带有配体的 AtASL 结构使我们能够详细分析酶与底物和酶与产物之间的相互作用。此外，根据我们的分析，我们描述了AtASL中对催化作用至关重要的残基。AtASL 的结构给出了 GSS 移动环从打开到关闭转变的基本原理，并指出了该环中的丝氨酸 333 对该酶的酶促作用的重要性。最后，我们通过鉴定 ASL 的特征序列图案对结构数据进行了补充。
{"title":"Arabidopsis thaliana argininosuccinate lyase structure uncovers the role of serine as the catalytic base","authors":"Maciej Nielipinski, Dominika Nielipinska, Agnieszka J. Pietrzyk-Brzezinska, Bartosz Sekula","doi":"10.1016/j.jsb.2024.108130","DOIUrl":"10.1016/j.jsb.2024.108130","url":null,"abstract":"<div><div>Arginine is an important amino acid in plants, as it not only plays a structural role and serves as nitrogen storage but is also a precursor for various molecules, including polyamines and proline. Arginine is produced by argininosuccinate lyase (ASL) which catalyzes the cleavage of argininosuccinate to arginine and fumarate. ASL belongs to the fumarate lyase family and while many members of this family were well-characterized, little is known about plant ASLs. Here we present the first crystal structures of ASL from the model plant, <em>Arabidopsis thaliana</em> (<em>At</em>ASL). One of the structures represents the unliganded form of the <em>At</em>ASL homotetramer. The other structure, obtained from a crystal soaked in argininosuccinate, accommodates the substrate or the reaction products in one of four active sites of the <em>At</em>ASL tetramer. Each active site is located at the interface of three neighboring protomers. The <em>At</em>ASL structure with ligands allowed us to analyze the enzyme-substrate and the enzyme-product interactions in detail. Furthermore, based on our analyses, we describe residues of <em>At</em>ASL crucial for catalysis. The structure of <em>At</em>ASL gives the rationale for the open-to-close transition of the GSS mobile loop and indicates the importance of serine 333 from this loop for the enzymatic action of the enzyme. Finally, we supplemented the structural data with the identification of sequence motifs characteristic for ASLs.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108130"},"PeriodicalIF":3.0,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.jsb.2024.108134
Yinghua Chen, Cassandra Villani, Amudha Ganapathy, Anne George
Dentin phosphophoryn (DPP), synthesized and processed predominantly by the odontoblasts, serves both a structural and signaling role in dentin. In the ECM, DPP functions as an avid calcium and collagen binding protein and it also plays a crucial role as a scaffold for cell attachment and survival. The signaling function of DPP was demonstrated when undifferentiated mesenchymal cells stimulated with DPP, mediated calcium signaling through release of intracellular Ca2+. The objective of this study was to identify potentially novel signaling mechanisms that mediate odontoblast differentiation. Therefore, transcriptomes of DPSCs (dental pulp stem cells) with or without DPP stimulation were compared by bulk RNA-seq. Analysis of the unbiased RNA-seq data were subjected to functional enrichment analysis using Gene Ontology (GO) and KEGG pathways. Results identified several upregulated genes which were associated with autophagy, that were subsequently validated by RT-PCR. Western blotting analysis confirmed the up regulation of several autophagy markers such as ATG5, BECN1 and LC3A/B at specific time points. Autophagosome formation was also observed with DPP treatment. Additionally, autophagy supported a role for odontoblast differentiation of DPSCs. These findings suggest that DPP mediated autophagy might be a potential mechanism for the survival and terminal differentiation of DPSCs.
{"title":"Transcriptome profiling of DPP stimulated DPSCs identifies the role of autophagy in odontogenic differentiation","authors":"Yinghua Chen, Cassandra Villani, Amudha Ganapathy, Anne George","doi":"10.1016/j.jsb.2024.108134","DOIUrl":"10.1016/j.jsb.2024.108134","url":null,"abstract":"<div><div>Dentin phosphophoryn (DPP), synthesized and processed predominantly by the odontoblasts, serves both a structural and signaling role in dentin. In the ECM, DPP functions as an avid calcium and collagen binding protein and it also plays a crucial role as a scaffold for cell attachment and survival. The signaling function of DPP was demonstrated when undifferentiated mesenchymal cells stimulated with DPP, mediated calcium signaling through release of intracellular Ca<sup>2+</sup>. The objective of this study was to identify potentially novel signaling mechanisms that mediate odontoblast differentiation. Therefore, transcriptomes of DPSCs (dental pulp stem cells) with or without DPP stimulation were compared by bulk RNA-seq. Analysis of the unbiased RNA-seq data were subjected to functional enrichment analysis using Gene Ontology (GO) and KEGG pathways. Results identified several upregulated genes which were associated with autophagy, that were subsequently validated by RT-PCR. Western blotting analysis confirmed the up regulation of several autophagy markers such as ATG5, BECN1 and LC3A/B at specific time points. Autophagosome formation was also observed with DPP treatment. Additionally, autophagy supported a role for odontoblast differentiation of DPSCs. These findings suggest that DPP mediated autophagy might be a potential mechanism for the survival and terminal differentiation of DPSCs.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108134"},"PeriodicalIF":3.0,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.jsb.2024.108133
Cassandra Villani, Yinghua Chen, Anne George
The oral microbiome dysbiosis that causes periodontal disease leads to disruption of various signaling pathways that can result in alveolar bone degradation and subsequent tooth loss. Previous studies have demonstrated the potential of stem cell-based therapies in regeneration of the lost periodontium for the preservation of natural dentition. Periodontal ligament stem cells (PDLSCs) have osteoblast differentiation potential and their proximity to bone makes them an ideal candidate for regenerative therapies. Dentin matrix protein 1 (DMP1), a non-collagenous extracellular matrix protein, is integral to mineralized tissue formation due to its dual roles as an extracellular mediator of hydroxyapatite deposition and intracellular regulator of osteoblastogenesis. Heat shock protein 5A (GRP78) is a master regulator of the endoplasmic reticulum stress response and previous studies in our laboratory have also demonstrated its function as a membrane receptor for DMP1. Bulk RNA sequencing analysis of PDLSCs and PDLSCs overexpressing GRP78 (PDLSCs GRP78) with or without treatment with DMP1 was conducted to evaluate alterations to the differentially expressed gene profiles. This study aims to elucidate pathways in PDLSCs that are altered upon treatment with DMP1 to further characterize its relationship with GRP78 and cell stress signaling cascades. Pathway enrichment analysis of each transcriptomic profile demonstrated enrichment of osteogenic and immune response pathways upon DMP1 stimulation. Results from this study indicate a novel role for DMP1 and GRP78 in modulating immune signaling cascades in PDLSCs.
{"title":"Role of DMP1-mediated GRP78 activation in osteoimmunomodulation of periodontal ligament stem cells","authors":"Cassandra Villani, Yinghua Chen, Anne George","doi":"10.1016/j.jsb.2024.108133","DOIUrl":"10.1016/j.jsb.2024.108133","url":null,"abstract":"<div><div>The oral microbiome dysbiosis that causes periodontal disease leads to disruption of various signaling pathways that can result in alveolar bone degradation and subsequent tooth loss. Previous studies have demonstrated the potential of stem cell-based therapies in regeneration of the lost periodontium for the preservation of natural dentition. Periodontal ligament stem cells (PDLSCs) have osteoblast differentiation potential and their proximity to bone makes them an ideal candidate for regenerative therapies. Dentin matrix protein 1 (DMP1), a non-collagenous extracellular matrix protein, is integral to mineralized tissue formation due to its dual roles as an extracellular mediator of hydroxyapatite deposition and intracellular regulator of osteoblastogenesis. Heat shock protein 5A (GRP78) is a master regulator of the endoplasmic reticulum stress response and previous studies in our laboratory have also demonstrated its function as a membrane receptor for DMP1. Bulk RNA sequencing analysis of PDLSCs and PDLSCs overexpressing GRP78 (PDLSCs <em>GRP78</em>) with or without treatment with DMP1 was conducted to evaluate alterations to the differentially expressed gene profiles. This study aims to elucidate pathways in PDLSCs that are altered upon treatment with DMP1 to further characterize its relationship with GRP78 and cell stress signaling cascades. Pathway enrichment analysis of each transcriptomic profile demonstrated enrichment of osteogenic and immune response pathways upon DMP1 stimulation. Results from this study indicate a novel role for DMP1 and GRP78 in modulating immune signaling cascades in PDLSCs.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108133"},"PeriodicalIF":3.0,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-05DOI: 10.1016/j.jsb.2024.108132
B.L. Foster
Bone sialoprotein (BSP) is a multi-functional extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. The amino acid sequence of BSP includes three evolutionarily conserved sequences which contribute to functions of the protein: an N-terminal collagen-binding domain, polyglutamic acid (polyE) sequences involved in hydroxyapatite nucleation and crystal growth, and a C-terminal arginine-glycine-aspartic acid (RGD) integrin-binding domain. BSP promotes attachment and differentiation of osteogenic and osteoclastic cells. Genetic ablation of BSP in mice results in skeletal and dental developmental defects and impaired bone healing in both appendicular bone and alveolar bone of the jaw.
Several studies demonstrated positive effects of BSP on bone healing in rodent models, though other experiments show negligible results. Native (harvested from rat bones) BSP cross-linked to collagen induced slight improvements in calvarial bone healing in rats. Recombinant BSP and collagen delivered in a polylactide (PLA) cylinder improved bone defect healing in rat femurs. Both native and recombinant BSP delivered in a collagen gel improved alveolar bone healing in wild-type and BSP-deficient mice. These advances suggest BSP is a new player in bone healing that has potential to be an alternative or complimentary to other bioactive factors. Future studies are necessary to understand mechanisms of how BSP influences bone healing and optimize delivery and dose in different types of bone defects and injuries.
{"title":"The role of bone sialoprotein in bone healing","authors":"B.L. Foster","doi":"10.1016/j.jsb.2024.108132","DOIUrl":"10.1016/j.jsb.2024.108132","url":null,"abstract":"<div><div>Bone sialoprotein (BSP) is a multi-functional extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. The amino acid sequence of BSP includes three evolutionarily conserved sequences which contribute to functions of the protein: an N-terminal collagen-binding domain, polyglutamic acid (polyE) sequences involved in hydroxyapatite nucleation and crystal growth, and a C-terminal arginine-glycine-aspartic acid (RGD) integrin-binding domain. BSP promotes attachment and differentiation of osteogenic and osteoclastic cells. Genetic ablation of BSP in mice results in skeletal and dental developmental defects and impaired bone healing in both appendicular bone and alveolar bone of the jaw.</div><div>Several studies demonstrated positive effects of BSP on bone healing in rodent models, though other experiments show negligible results. Native (harvested from rat bones) BSP cross-linked to collagen induced slight improvements in calvarial bone healing in rats. Recombinant BSP and collagen delivered in a polylactide (PLA) cylinder improved bone defect healing in rat femurs. Both native and recombinant BSP delivered in a collagen gel improved alveolar bone healing in wild-type and BSP-deficient mice. These advances suggest BSP is a new player in bone healing that has potential to be an alternative or complimentary to other bioactive factors. Future studies are necessary to understand mechanisms of how BSP influences bone healing and optimize delivery and dose in different types of bone defects and injuries.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108132"},"PeriodicalIF":3.0,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142381098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-04DOI: 10.1016/j.jsb.2024.108131
Sebastian T. Mergelsberg , Hoshin Kim , Garry W. Buchko , Bojana Ginovska
Amelogenin is an intrinsically disordered protein essential to tooth enamel formation in mammals. Using advanced small angle X-ray scattering (SAXS) capabilities at synchrotrons and computational models, we revisited measuring the quaternary structure of murine amelogenin as a function of pH and phosphorylation at serine-16. The SAXS data shows that at the pH extremes, amelogenin exists as an extended monomer at pH 3.0 (Rg = 38.4 Å) and nanospheres at pH 8.0 (Rg = 84.0 Å), consistent with multiple previous observations. At pH 5.0 and above there was no evidence for a significant population of monomeric species. Instead, at pH 5.0, ∼80 % of the population is a heterogenous dimeric species that increases to ∼100 % at pH 5.5. The dimer population was observed at all pH > 5 conditions in dynamic equilibrium with a species in the pentamer range at pH < 6.5 and nanospheres at pH 8.0. At pH 8.0, ∼40 % of the amelogenin remained in the dimeric state. In general, serine-16 phosphorylation of amelogenin appears to modestly stabilize the population of the dimeric species.
{"title":"SAXS of murine amelogenin identifies a persistent dimeric species from pH 5.0 to 8.0","authors":"Sebastian T. Mergelsberg , Hoshin Kim , Garry W. Buchko , Bojana Ginovska","doi":"10.1016/j.jsb.2024.108131","DOIUrl":"10.1016/j.jsb.2024.108131","url":null,"abstract":"<div><div>Amelogenin is an intrinsically disordered protein essential to tooth enamel formation in mammals. Using advanced small angle X-ray scattering (SAXS) capabilities at synchrotrons and computational models, we revisited measuring the quaternary structure of murine amelogenin as a function of pH and phosphorylation at serine-16. The SAXS data shows that at the pH extremes, amelogenin exists as an extended monomer at pH 3.0 (R<sub>g</sub> = 38.4 Å) and nanospheres at pH 8.0 (R<sub>g</sub> = 84.0 Å), consistent with multiple previous observations. At pH 5.0 and above there was no evidence for a significant population of monomeric species. Instead, at pH 5.0, ∼80 % of the population is a heterogenous dimeric species that increases to ∼100 % at pH 5.5. The dimer population was observed at all pH > 5 conditions in dynamic equilibrium with a species in the pentamer range at pH < 6.5 and nanospheres at pH 8.0. At pH 8.0, ∼40 % of the amelogenin remained in the dimeric state. In general, serine-16 phosphorylation of amelogenin appears to modestly stabilize the population of the dimeric species.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108131"},"PeriodicalIF":3.0,"publicationDate":"2024-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142378031","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The AXXXA and GXXXG motifs are frequently observed in helices, especially in membrane proteins. The motif GXXXG is known to stabilize helix-helix association in membrane proteins via CαHO bonding. AXXXA sequence motif additionally stabilizes the folded state of proteins. We found 27,000 and 18,000 occurrences of AXXXA and GXXXG motifs in a non-redundant set of 6000 obligate homodimeric (OD) complexes. Interestingly, this is less pronounced in transient homodimers (TD) and heterodimers (HetD). On average each obligate homodimer contains four AXXXA motifs, it is 2 and 3.5 for HetD and TD, respectively. Focusing on the binding surface it is seen that 27 % of the ODs contain at least one AXXXA motif at the interface, whereas it is 17 % and 15 % for HetD and TD respectively. AXXXA predominantly stabilizes the OD quaternary structure via the side chain CβCβ interactions. This interaction is energetically favorable and is found to be a major driving force for OD quaternary structure stability. Cβ-Cβ interactions are observed ∼6 times higher than the known CαHO interaction for helix-helix stabilization. Two additional new interactions of CβO and OO are observed at the AXXXA containing interface regions. The occurrence of the motif gets drastically reduced if any of the terminal Ala residues are replaced by Gly. Our findings show the importance of AXXXA in providing stability to the quaternary structure through specific hydrophobic interactions and the specificity of the Ala residue at motif termini. The knowledge gained can be used for designing synthetic proteins of improved stability and for designing peptide-based therapeutics.
AXXXA 和 GXXXG 主题经常在螺旋中出现，尤其是在膜蛋白中。已知 GXXXG 主题通过 CHO 键稳定膜蛋白中的螺旋-螺旋结合。此外，AXXXA 序列主题还能稳定蛋白质的折叠状态。我们发现，在一组非冗余的 6000 个强制性同源二聚体（OD）复合物中，AXXXA 和 GXXXG 主题分别出现了 27,000 和 18,000 次。有趣的是，这种情况在瞬时同源二聚体（TD）和异源二聚体（HetD）中并不明显。每个同源二聚体平均含有四个 AXXXA 矩阵，HetD 和 TD 分别为 2 个和 3.5 个。从结合表面可以看出，27%的 OD 在界面上至少含有一个 AXXXA 基序，而 HetD 和 TD 的这一比例分别为 17% 和 15%。AXXXA 主要通过侧链 CβCβ 相互作用来稳定 OD 的四元结构。这种相互作用在能量上是有利的，是 OD 四元结构稳定性的主要驱动力。观察到的 Cβ-Cβ 相互作用比已知的用于稳定螺旋-螺旋的 CHO 相互作用高出 6 倍。在含有 AXXXA 的界面区域还观察到 CβO 和 OO 的两种新的相互作用。如果任何一个末端的 Ala 残基被 Gly 取代，则该图案的出现率会急剧下降。我们的研究结果表明了 AXXXA 在通过特异性疏水相互作用为四元结构提供稳定性方面的重要性，以及图案末端 Ala 残基的特异性。所获得的知识可用于设计稳定性更好的合成蛋白质和设计基于肽的疗法。
{"title":"On the abundance and importance of AXXXA sequence motifs in globular proteins and their involvement in CβCβ interaction","authors":"Surbhi Vilas Tajane , Abhilasha Thakur , Srijita Acharya , Pinak Chakrabarti , Sucharita Dey","doi":"10.1016/j.jsb.2024.108129","DOIUrl":"10.1016/j.jsb.2024.108129","url":null,"abstract":"<div><div>The AXXXA and GXXXG motifs are frequently observed in helices, especially in membrane proteins. The motif GXXXG is known to stabilize helix-helix association in membrane proteins via C<sub>α</sub>H<img>O bonding. AXXXA sequence motif additionally stabilizes the folded state of proteins. We found 27,000 and 18,000 occurrences of AXXXA and GXXXG motifs in a non-redundant set of 6000 obligate homodimeric (OD) complexes. Interestingly, this is less pronounced in transient homodimers (TD) and heterodimers (HetD). On average each obligate homodimer contains four AXXXA motifs, it is 2 and 3.5 for HetD and TD, respectively. Focusing on the binding surface it is seen that 27 % of the ODs contain at least one AXXXA motif at the interface, whereas it is 17 % and 15 % for HetD and TD respectively. AXXXA predominantly stabilizes the OD quaternary structure via the side chain C<sub>β</sub><img>C<sub>β</sub> interactions. This interaction is energetically favorable and is found to be a major driving force for OD quaternary structure stability. C<sub>β-</sub>C<sub>β</sub> interactions are observed ∼6 times higher than the known C<sub>α</sub>H<img>O interaction for helix-helix stabilization. Two additional new interactions of C<sub>β</sub><img>O and O<img>O are observed at the AXXXA containing interface regions. The occurrence of the motif gets drastically reduced if any of the terminal Ala residues are replaced by Gly. Our findings show the importance of AXXXA in providing stability to the quaternary structure through specific hydrophobic interactions and the specificity of the Ala residue at motif termini. The knowledge gained can be used for designing synthetic proteins of improved stability and for designing peptide-based therapeutics.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108129"},"PeriodicalIF":3.0,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142349012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1016/j.jsb.2024.108128
Nadejda B. Matsko , Martin Schorb , Yannick Schwab
We present a Fourier transform (FT) based analytical method that allows to obtain of ultrastructural details from TEM images at sub-nanometer scale applying a selective filtering for singular macromolecule electron microscopy density information. It can be applied to high-pressure frozen, frozen hydrated and epoxy freeze substituted and embedded biological species. Both 2D projections and orthoslices from reconstructed tomograms can be used as a source of structural information. The key to the method is to select the macromolecule or organelle of interest with an accuracy of ≥ 7 – 3 nm (depending on pixel size of initial tilt series or singular image acquisition) and explore both the central low frequency FT intensity and diffraction regions to obtain the spatial structural organization and its dimensional characteristics, respectively. We also introduce a structure-specific selective mask FT filtering approach that can significantly improve image information even in poorly contrasted TEM of resin sections without heavy metal been used. The described method elucidates chromatin architecture without the need of averaging. A zigzag symmetry of 30 nm diameter chromatin fibers which in general is a controversial topic of research has been identified for C. elegans cells in vivo with sub-nanometer details being preserved in the images.
我们提出了一种基于傅立叶变换（FT）的分析方法，通过对奇异的大分子电子显微镜密度信息进行选择性过滤，可以从亚纳米尺度的 TEM 图像中获得超微结构细节。它适用于高压冷冻、冷冻水合和环氧树脂冷冻替代和嵌入的生物物种。二维投影和来自重建断层扫描的正切片都可用作结构信息的来源。该方法的关键在于选择感兴趣的大分子或细胞器，精确度≥ 7 - 3 nm（取决于初始倾斜序列或奇异图像采集的像素大小），并探索中心低频 FT 强度和衍射区域，以分别获得空间结构组织及其尺寸特征。我们还介绍了一种针对特定结构的选择性掩膜 FT 滤波方法，即使在对比度较差的树脂切片 TEM 中也能显著改善图像信息，而无需使用重金属。所述方法无需平均值即可阐明染色质结构。研究人员在体内发现了直径为 30 nm 的染色质纤维呈人字形对称，而这种染色质纤维在图像中保留了亚纳米级的细节。
{"title":"Selective signal enhancement in Fourier space as a tool for discovering ultrastructural organization of macromolecules from in situ TEM","authors":"Nadejda B. Matsko , Martin Schorb , Yannick Schwab","doi":"10.1016/j.jsb.2024.108128","DOIUrl":"10.1016/j.jsb.2024.108128","url":null,"abstract":"<div><div>We present a Fourier transform (FT) based analytical method that allows to obtain of ultrastructural details from TEM images at sub-nanometer scale applying a selective filtering for singular macromolecule electron microscopy density information. It can be applied to high-pressure frozen, frozen hydrated and epoxy freeze substituted and embedded biological species. Both 2D projections and orthoslices from reconstructed tomograms can be used as a source of structural information. The key to the method is to select the macromolecule or organelle of interest with an accuracy of ≥ 7 – 3 nm (depending on pixel size of initial tilt series or singular image acquisition) and explore both the central low frequency FT intensity and diffraction regions to obtain the spatial structural organization and its dimensional characteristics, respectively. We also introduce a structure-specific selective mask FT filtering approach that can significantly improve image information even in poorly contrasted TEM of resin sections without heavy metal been used. The described method elucidates chromatin architecture without the need of averaging. A zigzag symmetry of 30 nm diameter chromatin fibers which in general is a controversial topic of research has been identified for <em>C. elegans</em> cells <em>in vivo</em> with sub-nanometer details being preserved in the images.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108128"},"PeriodicalIF":3.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Trichomonas vaginalis is a parasite protozoan that causes human trichomoniasis, a sexually transmitted infection (STI) that affects more than 156 million people worldwide. T. vaginalis contains an uncommon and complex cytoskeleton constituting the mastigont system, formed by several fibers and proteinaceous structures associated with basal bodies. Among these structures is the pelta-axostylar complex made of microtubules and striated filaments such as the costa and the parabasal filaments. In addition, some structures are poorly known and studied, such as the sigmoid filament and the X-filament. Here, we have isolated the Trichomonas vaginalis cytoskeleton and used UHR-SEM (ultra-high resolution scanning electron microscopy), tomography, immunofluorescence, immunolabeling, and backscattered electrons on SEM, negative staining to model the three-dimensional architecture and possible function of the sigmoid.
阴道毛滴虫（Trichomonas vaginalis）是一种原生寄生虫，可导致人类滴虫病，这是一种性传播感染（STI），全球有超过 1.56 亿人受到感染。阴道毛滴虫含有一种不常见的复杂细胞骨架，由与基底体相关的几种纤维和蛋白结构组成，构成马氏体系统。在这些结构中，有由微管和条状细丝（如costa和parabasal细丝）组成的pelta-axostylar复合体。此外，对一些结构的了解和研究也不多，如乙状丝和 X 丝。在这里，我们分离了阴道毛滴虫的细胞骨架，并利用超高分辨率扫描电子显微镜（UHR-SEM）、断层扫描、免疫荧光、免疫标记和扫描电子显微镜上的背散射电子、阴性染色来模拟乙状结肠的三维结构和可能的功能。
{"title":"Three-dimensional cellular architecture of the sigmoid filament in Trichomonas vaginalis","authors":"Sharmila Ortiz , Raphael Verdan , Marlene Benchimol","doi":"10.1016/j.jsb.2024.108127","DOIUrl":"10.1016/j.jsb.2024.108127","url":null,"abstract":"<div><p><em>Trichomonas vaginalis</em> is a parasite protozoan that causes human trichomoniasis, a sexually transmitted infection (STI) that affects more than 156 million people worldwide. <em>T. vaginalis</em> contains an uncommon and complex cytoskeleton constituting the mastigont system, formed by several fibers and proteinaceous structures associated with basal bodies. Among these structures is the pelta-axostylar complex made of microtubules and striated filaments such as the costa and the parabasal filaments. In addition, some structures are poorly known and studied, such as the sigmoid filament and the X-filament. Here, we have isolated the <em>Trichomonas vaginalis</em> cytoskeleton and used UHR-SEM (ultra-high resolution scanning electron microscopy), tomography, immunofluorescence, immunolabeling, and backscattered electrons on SEM, negative staining to model the three-dimensional architecture and possible function of the sigmoid.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108127"},"PeriodicalIF":3.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145839","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The global crystallographic texture of calcite and aragonite in the shells of the bivalves Bathymodiolus thermophilus, Mytilus galloprovincialis, M. edulis and M. trossulus was studied by means of neutron diffraction. It was revealed that the general appearance of pole figures isolines of both minerals coincides for the studied species. The crystallographic texture sharpness evaluated by means of pole density on the calcite pole figures ((0006), ) and aragonite pole figures ((012)/(121), (040)/(221)) coincides or has close values for deep-sea hydrothermal species B. thermophilus and the studied shallow-water species of the genus Mytilus. The calcite pole figures (0006) and of B. thermophilus show a shift in the position of texture maximum values compared to corresponding pole figures of other mussels. The shell microstructure of all studied mollusks is similar, only the shape of the fibers of B. thermophilus differs. Global crystallographic texture is a stable feature of the family Mytilidae. The extreme habitat conditions of the hydrothermal biotope do not significantly affect the crystallographic texture of B. thermophilus.
{"title":"Comparison of the global crystallographic texture of minerals in the shells of Bathymodiolus thermophilus Kenk et B.R. Wilson, 1985 and species of the genus Mytilus Linnaeus, 1758","authors":"Alexey Pakhnevich , Dmitry Nikolayev , Tatiana Lychagina","doi":"10.1016/j.jsb.2024.108126","DOIUrl":"10.1016/j.jsb.2024.108126","url":null,"abstract":"<div><p>The global crystallographic texture of calcite and aragonite in the shells of the bivalves <em>Bathymodiolus thermophilus</em>, <em>Mytilus galloprovincialis</em>, <em>M. edulis</em> and <em>M. trossulus</em> was studied by means of neutron diffraction. It was revealed that the general appearance of pole figures isolines of both minerals coincides for the studied species. The crystallographic texture sharpness evaluated by means of pole density on the calcite pole figures ((0006), <span><math><mrow><mo>(</mo><mn>10</mn><mover><mrow><mn>1</mn></mrow><mrow><mo>¯</mo></mrow></mover><mn>4</mn><mo>)</mo></mrow></math></span>) and aragonite pole figures ((012)/(121), (040)/(221)) coincides or has close values for deep-sea hydrothermal species <em>B. thermophilus</em> and the studied shallow-water species of the genus <em>Mytilus</em>. The calcite pole figures (0006) and <span><math><mrow><mo>(</mo><mn>10</mn><mover><mrow><mn>1</mn></mrow><mrow><mo>¯</mo></mrow></mover><mn>4</mn><mo>)</mo></mrow></math></span> of <em>B. thermophilus</em> show a shift in the position of texture maximum values compared to corresponding pole figures of other mussels. The shell microstructure of all studied mollusks is similar, only the shape of the fibers of <em>B. thermophilus</em> differs. Global crystallographic texture is a stable feature of the family Mytilidae. The extreme habitat conditions of the hydrothermal biotope do not significantly affect the crystallographic texture of <em>B. thermophilus</em>.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 4","pages":"Article 108126"},"PeriodicalIF":3.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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