Septins belong to a family of proteins that is highly conserved in eukaryotes and is increasingly recognized as a novel component of the cytoskeleton. All septins are GTP-binding proteins that form hetero-oligomeric complexes and higher-order structures, including filaments and rings. Recent studies have provided structural information about the different levels of septin organization; however, the crucial structural determinants and factors responsible for septin assembly remain unclear. Investigations on the molecular functions of septins have highlighted their roles as scaffolds for protein recruitment and as diffusion barriers for subcellular compartmentalization in numerous biological processes, including cell division and host-microorganism interactions.

Septins: the fourth component of the cytoskeleton

Septins comprise a conserved family of proteins that are found primarily in fungi and animals. These GTP-binding proteins have several roles during cell division, cytoskeletal organization and membrane-remodelling events. One factor that is crucial for their functions is the ordered assembly of individual septins into oligomeric core complexes that, in turn, form higher-order structures such as filaments, rings and gauzes. The molecular details of these interactions and the mechanism by which septin-complex assembly is regulated have remained elusive. Recently, the first detailed structural views of the septin core have emerged, and these, along with studies of septin dynamics in vivo, have provided new insight into septin-complex assembly and septin function in vivo.

The septin family of GTPases: architecture and dynamics.

Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7) Only Shs1 is nonessential The four essential septins form a complex containing two copies of each, but their arrangement was not known Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein) The rod has the order Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 and, hence, lacks polarity At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered Filaments invariably pair into long parallel “railroad tracks” Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament Shs1 may be able to replace Cdc11 at the end of the rod Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures

https://www.pnas.org/content/pnas/105/24/8274.full.pdf

Saccharomyces cerevisiae septins: Supramolecular organization of heterooligomers and the mechanism of filament assembly

https://www.cell.com/developmental-cell/pdf/S1534-5807(09)00139-7.pdf

Septins and the lateral compartmentalization of eukaryotic membranes.

Background: Metazoan multicellularity is rooted in mechanisms of cell adhesion, signaling, and differentiation that first evolved in the progenitors of metazoans. To reconstruct the genome composition of metazoan ancestors, we sequenced the genome and transcriptome of the choanoflagellate Salpingoeca rosetta, a close relative of metazoans that forms rosette-shaped colonies of cells. Results: A comparison of the 55 Mb S. rosetta genome with genomes from diverse opisthokonts suggests that the origin of metazoans was preceded by a period of dynamic gene gain and loss. The S. rosetta genome encodes homologs of cell adhesion, neuropeptide, and glycosphingolipid metabolism genes previously found only in metazoans and expands the repertoire of genes inferred to have been present in the progenitors of metazoans and choanoflagellates. Transcriptome analysis revealed that all four S. rosetta septins are upregulated in colonies relative to single cells, suggesting that these conserved cytokinesis proteins may regulate incomplete cytokinesis during colony development. Furthermore, genes shared exclusively by metazoans and choanoflagellates were disproportionately upregulated in colonies and the single cells from which they develop. Conclusions: The S. rosetta genome sequence refines the catalog of metazoan-specific genes while also extending the evolutionary history of certain gene families that are central to metazoan biology. Transcriptome data suggest that conserved cytokinesis genes, including septins, may contribute to S. rosetta colony formation and indicate that the initiation of colony development may preferentially draw upon genes shared with metazoans, while later stages of colony maturation are likely regulated by genes unique to S. rosetta. Background Metazoan multicellularity and development are rooted in basic mechanisms of cell adhesion, signaling, and differentiation that were present in the unicellular and colonial progenitors of metazoans. Reconstructing the evolution of metazoans from their single celled ancestors promises to illuminate one of the major transitions in evolutionary history, while also revealing fundamental mechanisms underlying metazoan cell biology and

/pdf/premetazoan-genome-evolution-and-the-regulation-of-cell-1abe4lz5xv.pdf

Premetazoan genome evolution and the regulation of cell differentiation in the choanoflagellate Salpingoeca rosetta

Septins are cytoskeletal GTPase proteins first discovered in the fungus Saccharomyces cerevisiae where they organize the septum and link nuclear division with cell division. More recently septins have been found in animals where they are important in processes ranging from actin and microtubule organization to embryonic patterning and where defects in septins have been implicated in human disease. Previous studies suggested that many animal septins fell into independent evolutionary groups, confounding cross-kingdom comparison. In the current work, we identified 162 septins from fungi, microsporidia and animals and analyzed their phylogenetic relationships. There was support for five groups of septins with orthology between kingdoms. Group 1 (which includes S. cerevisiae Cdc10p and human Sept9) and Group 2 (which includes S. cerevisiae Cdc3p and human Sept7) contain sequences from fungi and animals. Group 3 (which includes S. cerevisiae Cdc11p) and Group 4 (which includes S. cerevisiae Cdc12p) contain sequences from fungi and microsporidia. Group 5 (which includes Aspergillus nidulans AspE) contains sequences from filamentous fungi. We suggest a modified nomenclature based on these phylogenetic relationships. Comparative sequence alignments revealed septin derivatives of already known G1, G3 and G4 GTPase motifs, four new motifs from two to twelve amino acids long and six conserved single amino acid positions. One of these new motifs is septin-specific and several are group specific. Our studies provide an evolutionary history for this important family of proteins and a framework and consistent nomenclature for comparison of septin orthologs across kingdoms.

/pdf/analysis-of-septins-across-kingdoms-reveals-orthology-and-110l9jthnp.pdf

Analysis of septins across kingdoms reveals orthology and new motifs

Evolution and Conserved Domains of the Septins

RATIONALE
Hydroxychloroquine sulfate is effective in the treatment of malaria, autoimmune diseases, and as an antiviral drug. However, unreported impurities are often detected in this drug, which pose a health risk. In this study, the structures of hydroxychloroquine and six unknown impurities were analyzed using ultra-high performance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry (UHPLC-Q/TOF MS), and the structures were characterized using liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy (LC-SPE-NMR).


METHODS
The column was an Agilent InfinityLad Poroshell HPH-C18 (100 mm × 4.6 mm, 2.7 μm). For the analysis of hydroxychloroquine and six unknown impurities, the mobile phase was 20 mM ammonium formate aqueous solution and methanol/acetonitrile (80:20, v/v), using gradient elution. Full-scan MS and MS2 were performed in order to obtain as much structural information as possible. Additionally, six unknown impurities were separated by semi-preparative liquid chromatography and characterized by LC-SPE-NMR.


RESULTS
The MS2 fragmentation patterns of the impurities were investigated, leading to more structural information and an understanding of the fragmentation pathways of the impurities. The unknown impurities' structures were confirmed by NMR. In addition, some possible pathways of the formation of the impurities in the drugs were outlined, and these impurities were found to be process impurities.


CONCLUSIONS
Based on the identification and characterization of these impurities, this study also describes the cause of the production of the impurities and provides insights for companies to improve their production processes and a scientific basis for the improvement of the related pharmacopoeias.

A study of impurities in the repurposed COVID-19 drug hydroxychloroquine sulfate by UHPLC-Q/TOF-MS and LC-SPE-NMR.

Rationale Hydroxychloroquine sulfate is effective in the treatment of malaria and autoimmune diseases and as an antiviral drug. However, unreported impurities are often detected in this drug, which pose a health risk. In this study, the structures of hydroxychloroquine and six unknown impurities were analyzed using ultra‐high‐performance liquid chromatography‐quadrupole/time‐of‐flight‐tandem mass spectrometry (UHPLCQ/TOF/MS/MS), and the structures were characterized using liquid chromatography‐solid‐phase extraction‐nuclear magnetic resonance (LC‐SPE‐NMR) spectroscopy. Methods An Agilent InfinityLad Poroshell HPH‐C18 column (100 × 4.6 mm, 2.7 μm) was used. For the analysis of hydroxychloroquine and six unknown impurities, the mobile phase was 20 mM ammonium formate aqueous solution and methanol/acetonitrile (80:20, v/v) using gradient elution. Full‐scan MS and MS2 were performed to obtain as much structural information as possible. In addition, six unknown impurities were separated by semipreparative liquid chromatography and characterized using LC‐SPE‐NMR. Results The MS2 fragmentation patterns of the impurities were investigated, leading to more structural information and an understanding of the fragmentation pathways of the impurities. The structures of the unknown impurities were confirmed using NMR. In addition, some possible pathways of the formation of the impurities in the drugs were outlined, and these impurities were found to be process impurities. Conclusions Based on the identification and characterization of these impurities, this study also describes the cause of the production of the impurities and provides insights for companies to improve their production processes and a scientific basis for the improvement of the related pharmacopoeias.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9350102

A study of impurities in the repurposed COVID‐19 drug hydroxychloroquine sulfate using ultra‐high‐performance liquid chromatography‐quadrupole/time‐of‐flight mass spectrometry and liquid chromatography‐solid‐phase extraction‐nuclear magnetic resonance

Fangfang Pan

Papers

Analysis of septins across kingdoms reveals orthology and new motifs

Evolution and Conserved Domains of the Septins

A study of impurities in the repurposed COVID-19 drug hydroxychloroquine sulfate by UHPLC-Q/TOF-MS and LC-SPE-NMR.

A study of impurities in the repurposed COVID‐19 drug hydroxychloroquine sulfate using ultra‐high‐performance liquid chromatography‐quadrupole/time‐of‐flight mass spectrometry and liquid chromatography‐solid‐phase extraction‐nuclear magnetic resonance