New Insights on Structure and Function of Sialyltransferases

Sialic acids are a unique posttranslational modification at the terminus of glycoproteins and -lipids. Proteins modified with oligomers of sialic acid add a repellent charge to cell surfaces, which is a crucial feature in cell migration and axonal growth during early brain development. Varied expression levels of sialic acid are linked to tumor malignancy in neuroblastoma, schizophrenia, autism and bipolar disorder but the lack thereof is linked to impaired neuronal development. On the other hand, overexpression of sialic acid oligomers in Schwann cells promotes the peripheral regeneration of lesioned nerves and improves the ability of Schwann cells to migrate into damaged tissue and to remyelinate central nervous system axons. In order to understand the molecular mechanisms of sialylation, our project focuses on the structural characterization of enzymes of the mammalian and bacterial glycosyltransferase families 29 and 42. The proteins of interest were expressed in insect cells and structural studies were undertaken by x-ray crystallography. Kinetics, SEC MALS and glycan array data will shed light on mechanism of catalysis and acceptor specificity. Altogether, the results of this study will promote further understanding of the structure-function relationship of sialyltransferases.

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Heavy Atom Detergent/Lipid Combined X-ray Crystallography for Elucidating the Structure-Function Relationships of Membrane Proteins

Membranes ◽

10.3390/membranes11110823 ◽

2021 ◽

Vol 11 (11) ◽

pp. 823

Author(s):

Shinya Hanashima ◽

Takanori Nakane ◽

Eiichi Mizohata

Keyword(s):

Membrane Proteins ◽

Structure Function ◽

New Technologies ◽

Heavy Atom ◽

Protein Structures ◽

X Ray ◽

X Ray Crystallography ◽

Function Relationship ◽

And Function ◽

Relationship Of

Membrane proteins reside in the lipid bilayer of biomembranes and the structure and function of these proteins are closely related to their interactions with lipid molecules. Structural analyses of interactions between membrane proteins and lipids or detergents that constitute biological or artificial model membranes are important for understanding the functions and physicochemical properties of membrane proteins and biomembranes. Determination of membrane protein structures is much more difficult when compared with that of soluble proteins, but the development of various new technologies has accelerated the elucidation of the structure-function relationship of membrane proteins. This review summarizes the development of heavy atom derivative detergents and lipids that can be used for structural analysis of membrane proteins and their interactions with detergents/lipids, including their application with X-ray free-electron laser crystallography.

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Regeneration of Planarian Auricles and Reestablishment of Chemotactic Ability

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.777951 ◽

2021 ◽

Vol 9 ◽

Author(s):

Eugene Matthew P. Almazan ◽

Joseph F. Ryan ◽

Labib Rouhana

Keyword(s):

Molecular Mechanisms ◽

Behavioral Experiments ◽

X Ray ◽

Preferential Expression ◽

Chemical Stimuli ◽

And Function ◽

Function Models ◽

Chemotactic Ability

Detection of chemical stimuli is crucial for living systems and also contributes to quality of life in humans. Since loss of olfaction becomes more prevalent with aging, longer life expectancies have fueled interest in understanding the molecular mechanisms behind the development and maintenance of chemical sensing. Planarian flatworms possess an unsurpassed ability for stem cell-driven regeneration that allows them to restore any damaged or removed part of their bodies. This includes anteriorly-positioned lateral flaps known as auricles, which have long been thought to play a central role in chemotaxis. The contribution of auricles to the detection of positive chemical stimuli was tested in this study using Girardia dorotocephala, a North American planarian species known for its morphologically prominent auricles. Behavioral experiments staged under laboratory conditions revealed that removal of auricles by amputation leads to a significant decrease in the ability of planarians to find food. However, full chemotactic capacity is observed as early as 2 days post-amputation, which is days prior from restoration of auricle morphology, but correlative with accumulation of ciliated cells in the position of auricle regeneration. Planarians subjected to x-ray irradiation prior to auricle amputation were unable to restore auricle morphology, but were still able to restore chemotactic capacity. These results indicate that although regeneration of auricle morphology requires stem cells, some restoration of chemotactic ability can still be achieved in the absence of normal auricle morphology, corroborating with the initial observation that chemotactic success is reestablished 2-days post-amputation in our assays. Transcriptome profiles of excised auricles were obtained to facilitate molecular characterization of these structures, as well as the identification of genes that contribute to chemotaxis and auricle development. A significant overlap was found between genes with preferential expression in auricles of G. dorotocephala and genes with reduced expression upon SoxB1 knockdown in Schmidtea mediterranea, suggesting that SoxB1 has a conserved role in regulating auricle development and function. Models that distinguish between possible contributions to chemotactic behavior obtained from cellular composition, as compared to anatomical morphology of the auricles, are discussed.

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Structure and function relationship of phosphatidylglycerol in the stabilization of phosphatidylethanolamine bilayer

Biochemistry ◽

10.1021/bi00445a028 ◽

1989 ◽

Vol 28 (19) ◽

pp. 7708-7712 ◽

Cited By ~ 33

Author(s):

Ana Tari ◽

Leaf Huang

Keyword(s):

Structure And Function ◽

Function Relationship ◽

Structure And Function Relationship ◽

And Function ◽

Relationship Of

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Antiviral adhesion molecular mechanisms for influenza: W. G. Laver's lifetime obsession

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0034 ◽

2015 ◽

Vol 370 (1661) ◽

pp. 20140034 ◽

Cited By ~ 1

Author(s):

Elspeth F. Garman

Keyword(s):

Sialic Acid ◽

Host Cell ◽

Molecular Mechanisms ◽

Surface Protein ◽

X Ray Crystallography ◽

Sialic Acid Receptors ◽

Sialic Acid Binding ◽

Viral Surface Protein ◽

Viral Surface

Infection by the influenza virus depends firstly on cell adhesion via the sialic-acid-binding viral surface protein, haemagglutinin, and secondly on the successful escape of progeny viruses from the host cell to enable the virus to spread to other cells. To achieve the latter, influenza uses another glycoprotein, the enzyme neuraminidase (NA), to cleave the sialic acid receptors from the surface of the original host cell. This paper traces the development of anti-influenza drugs, from the initial suggestion by MacFarlane Burnet in 1948 that an effective ‘competitive poison’ of the virus' NA might be useful in controlling infection by the virus, through to the determination of the structure of NA by X-ray crystallography and the realization of Burnet's idea with the design of NA inhibitors. A focus is the contribution of the late William Graeme Laver, FRS, to this research.

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The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011277 ◽

2020 ◽

Vol 295 (9) ◽

pp. 2822-2838 ◽

Cited By ~ 3

Author(s):

Alexandra A. Richter ◽

Stefanie Kobus ◽

Laura Czech ◽

Astrid Hoeppner ◽

Jan Zarzycki ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Catalytic Core ◽

Chemical Chaperone ◽

Practical Applications ◽

Acetyl Group ◽

Function Relationship ◽

Thermotolerant Bacterium ◽

Stress Protectant ◽

Relationship Of

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2–2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

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Molecular mechanisms behind mRNA localization in axons

Open Biology ◽

10.1098/rsob.200177 ◽

2020 ◽

Vol 10 (9) ◽

pp. 200177

Author(s):

Benita Turner-Bridger ◽

Cinzia Caterino ◽

Jean-Michel Cioni

Keyword(s):

Messenger Rna ◽

Molecular Mechanisms ◽

Neuronal Development ◽

Mrna Localization ◽

Adaptive Responses ◽

Cis Acting ◽

Spatiotemporal Regulation ◽

Mrna Content ◽

Specific Mrnas ◽

And Function

Messenger RNA (mRNA) localization allows spatiotemporal regulation of the proteome at the subcellular level. This is observed in the axons of neurons, where mRNA localization is involved in regulating neuronal development and function by orchestrating rapid adaptive responses to extracellular cues and the maintenance of axonal homeostasis through local translation. Here, we provide an overview of the key findings that have broadened our knowledge regarding how specific mRNAs are trafficked and localize to axons. In particular, we review transcriptomic studies investigating mRNA content in axons and the molecular principles underpinning how these mRNAs arrived there, including cis-acting mRNA sequences and trans-acting proteins playing a role. Further, we discuss evidence that links defective axonal mRNA localization and pathological outcomes.

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Cytolethal Distending Toxin Subunit B: A Review of Structure–Function Relationship

Toxins ◽

10.3390/toxins11100595 ◽

2019 ◽

Vol 11 (10) ◽

pp. 595 ◽

Cited By ~ 5

Author(s):

Benoît J. Pons ◽

Julien Vignard ◽

Gladys Mirey

Keyword(s):

Pathogenic Bacteria ◽

Molecular Mechanisms ◽

3D Structure ◽

Cytolethal Distending Toxin ◽

Experimental Systems ◽

Bacterial Virulence Factor ◽

Function Relationship ◽

And Function ◽

Key Residues ◽

Cell Cycle Block

The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions.

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General discussion summary

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2002.1149 ◽

2002 ◽

Vol 357 (1426) ◽

pp. 1419-1420 ◽

Cited By ~ 2

Keyword(s):

Water Splitting ◽

Molecular Mechanisms ◽

Structure And Function ◽

X Ray ◽

X Ray Crystallography ◽

And Function ◽

Splitting Process

This general discussion was chaired by A. W. Rutherford ( Service de Bioénergétique, Saclay, France ) and revolved around two major topics: (i) the implications of X–ray crystallography on the relationships between structure and function; (ii) the molecular mechanisms of the water–splitting process.

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