Arabidopsis Carboxylesterase 20 Binds Strigolactone and Increases Branches and Tillers When Ectopically Expressed in Arabidopsis and Maize

Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.

Download Full-text

Structure of the Lifeact–F-actin complex

PLoS Biology ◽

10.1371/journal.pbio.3000925 ◽

2020 ◽

Vol 18 (11) ◽

pp. e3000925 ◽

Cited By ~ 1

Author(s):

Alexander Belyy ◽

Felipe Merino ◽

Oleg Sitsel ◽

Stefan Raunser

Keyword(s):

Hypervariable Region ◽

Binding Pocket ◽

Actin Binding ◽

Filamentous Actin ◽

Activity Measurements ◽

High Resolution Structure ◽

D Loop ◽

Hydrophobic Binding

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

Download Full-text

Characterization of calcium- and integrin-binding protein 1 (CIB1) knockout platelets: Potential compensation by CIB family members

Thrombosis and Haemostasis ◽

10.1160/th08-06-0351 ◽

2008 ◽

Vol 100 (05) ◽

pp. 847-856 ◽

Cited By ~ 19

Author(s):

Brenda R. Temple ◽

Holly R. Gentry ◽

Jan C. DeNofrio ◽

Weiping Yuan ◽

Leslie V. Parise

Keyword(s):

Thrombus Formation ◽

Mrna Level ◽

Family Members ◽

Cytoplasmic Tail ◽

Binding Pocket ◽

Vitro Binding ◽

Cytoplasmic Tails ◽

Hydrophobic Binding

SummaryPlatelet aggregation requires activation of the αIIbβ3 integrin,an event regulated by the integrin cytoplasmic tails. CIB1 binds to the cytoplasmic tail of the integrin αIIb subunit. Previous overexpression and knockdown studies in murine megakaryocytes demonstrated that CIB1 inhibits integrin αIIbβ3 activation.Here we analyzed Cib1-/- mice to determine the function of CIB1 in platelets in vitro and in vivo. We found that although these mice had no overt platelet phenotype, mRNA level of CIB1 homolog CIB3 was increased in Cib1-/- megakaryocytes. In vitro binding experiments showed that recombinant CIB1, -2 and -3 bound specifically to an αIIb cytoplasmic tail peptide. Subsequent protein modeling experiments indicated that CIBs 1–3 each have a highly conserved hydrophobic binding pocket. Therefore, the potential exists for compensation for the loss of CIB1 by these CIB family members, thereby preventing pathologic thrombus formation in Cib1-/- mice.

Download Full-text

Congenital Lipoid Adrenal Hyperplasia: Functional Characterization of Three Novel Mutations in the STAR Gene

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2009-1176 ◽

2010 ◽

Vol 95 (3) ◽

pp. 1301-1308 ◽

Cited By ~ 20

Author(s):

Susanne Bens ◽

Angelika Mohn ◽

Bilgin Yüksel ◽

Alexandra E. Kulle ◽

Matthias Michalek ◽

...

Keyword(s):

Functional Characterization ◽

Regulatory Protein ◽

Three Dimensional ◽

Binding Pocket ◽

Adrenal Hyperplasia ◽

Turkish Patient ◽

Star Protein ◽

Sex Development ◽

Congenital Lipoid Adrenal Hyperplasia

Abstract Context: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia. Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient). Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the α4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein. Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient’s phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

Download Full-text

Transglutaminase 5 is regulated by guanine–adenine nucleotides1

Biochemical Journal ◽

10.1042/bj20031474 ◽

2004 ◽

Vol 381 (1) ◽

pp. 313-319 ◽

Cited By ~ 35

Author(s):

Eleonora CANDI ◽

Andrea PARADISI ◽

Alessandro TERRINONI ◽

Valentina PIETRONI ◽

Sergio ODDI ◽

...

Keyword(s):

Amino Acid ◽

Three Dimensional ◽

Binding Pocket ◽

Cross Linking ◽

Bifunctional Enzyme ◽

Amino Acid Sequence Alignment ◽

Gtp Binding ◽

Epidermal Tissue ◽

Isopeptide Bonds

Transglutaminases (TGases) are Ca2+-dependent enzymes capable of catalysing transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGases have been described in mammals, and two of them (types 2 and 3) are regulated by GTP/ATP. TGase2 hydrolyses GTP and is therefore a bifunctional enzyme. In the present study, we report that TGase5 is also regulated by nucleotides. We have identified the putative TGase5 GTP-binding pocket by comparative amino acid sequence alignment and homology-derived three-dimensional modelling. GTP and ATP inhibit TGase5 cross-linking activity in vitro, and Ca2+ is capable of completely reversing this inhibition. In addition, TGase5 mRNA is not restricted to epidermal tissue, but is also present in different adult and foetal tissues, suggesting a role for TGase5 outside the epidermis. These results reveal the reciprocal actions of Ca2+ and nucleotides with respect to TGase5 activity. Taken together, these results indicate that TGases are a complex family of enzymes regulated by calcium, with at least three of them, namely TGase2, TGase3 and TGase5, also being regulated by ATP and GTP.

Download Full-text

Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis

Blood ◽

10.1182/blood-2009-04-218735 ◽

2010 ◽

Vol 115 (7) ◽

pp. 1444-1452 ◽

Cited By ~ 77

Author(s):

Céline Cougoule ◽

Véronique Le Cabec ◽

Renaud Poincloux ◽

Talal Al Saati ◽

Jean-Louis Mège ◽

...

Keyword(s):

Extracellular Matrix ◽

Spatial Organization ◽

Ectopic Expression ◽

Three Dimensional ◽

Matrix Metalloproteases ◽

3 Dimensional ◽

Normal Expression ◽

Migration Of Macrophages

Abstract Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck−/− mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow–derived macrophages (BMDMs), were affected in Hck−/− BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain–of–3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules.

Download Full-text

New approach for T-shaped uterus: Metroplasty with resection of lateral fibromuscular tissue using a 15 Fr miniresectoscope. A step-by-step technique.

Facts, Views and Vision in ObGyn ◽

10.52054/fvvo.13.003 ◽

2021 ◽

Vol 13 (1) ◽

pp. 67-71

Author(s):

U. Catena ◽

R. Campo ◽

G. Bolomini ◽

M.C. Moruzzi ◽

V. Verdecchia ◽

...

Keyword(s):

Surgical Technique ◽

Recurrent Miscarriage ◽

Three Dimensional ◽

Uterine Cavity ◽

In Vitro Fertilisation ◽

Step Method ◽

New Approach ◽

Uterine Malformation ◽

New Surgical Technique

T-shaped uterus is a congenital uterine malformation (CUM), only recently defined by the ESGE ESHRE classification as Class U1a. The uterus is characterised by a narrow uterine cavity due to thickened lateral walls with a correlation 2/3 uterine corpus and 1/3 cervix (Grimbizis et al, 2013). Although the significance of this dysmorphic malformation on reproductive performance has been questioned, recent studies reported significant improvement of life birth rates after surgical correction in patients with failed in-vitro fertilisation (IVF) or recurrent miscarriage (Ferro et al, 2018; Di Spiezio Sardo et al, 2020; Alonso Pacheco et al. 2019). The classical surgical technique to treat a T-shaped uterus is by performing a sidewall incision with the micro scissor or bipolar needle, resulting in a triangular cavity. In this video article, we describe a new surgical technique with a step-by-step method combining three-dimensional ultrasound (3D-US) and hysteroscopic metroplasty in an office setting, using a 15 Fr office resectoscope (Karl Storz, Tuttlingen, Germany), to treat a T-shaped uterus by resecting the lateral fibromuscular tissue of the uterine walls. No complications occurred and the postoperative hysteroscopy showed a triangular and symmetrical uterine cavity without any adhesions.

Download Full-text

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713026461 ◽

2014 ◽

Vol 70 (2) ◽

pp. 242-252 ◽

Cited By ~ 4

Author(s):

Sonia Fieulaine ◽

Michel Desmadril ◽

Thierry Meinnel ◽

Carmela Giglione

Keyword(s):

Sequence Similarity ◽

Three Dimensional ◽

Binding Pocket ◽

Dimensional Structure ◽

Structural Basis ◽

Human Counterpart ◽

Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

Download Full-text

Improved Understanding of Stent Malapposition Using Virtual Bench Testing

Interventional Cardiology Review ◽

10.15420/icr.2011.6.2.106 ◽

2011 ◽

Vol 6 (2) ◽

pp. 106 ◽

Cited By ~ 5

Author(s):

Peter Mortier ◽

Heleen MM van Beusekom ◽

Matthieu De Beule ◽

Ilona Krabbendam-Peters ◽

Benjamin Van Der Smissen ◽

...

Keyword(s):

Imaging Techniques ◽

Average Distance ◽

Three Dimensional ◽

Proximal Part ◽

New Approach ◽

Incomplete Stent Apposition ◽

Bifurcation Stenting ◽

Silicone Model ◽

Bench Testing

Intravascular imaging techniques such as optical coherence tomography (OCT) and intravascular ultrasound (IVUS) are often used to assess strut apposition, but only provide limited insight into the three-dimensional appositioning behaviour of stents. Recently, a new approach has been introduced to study the phenomenon of incomplete stent apposition (ISA) based on finite element simulations. In this study, we employed this virtual strut apposition assessment technique in the setting of coronary bifurcation stenting and compared simulated strut–artery distances of two stent designs with actual measurements based on OCT imaging using a silicone model. Stenting of the main branch leads to malapposed struts in the proximal part and the average strut–artery distance in that region for the Integrity stent is 126 μm based on the simulation and 117±14 μm based on the OCT analysis. For the Multi-Link 8 stent, this average distance is 150 μm and 174±7 µm for the simulation and thein vitroOCT measurements respectively. In conclusion, the virtual assessment of strut appositioning results in similar strut–artery distances when compared with measurements based on OCT-visualisedin vitrostent deployments and could be used to optimise devices and procedures.

Download Full-text

Biochemical and three-dimensional-structural study of the specific inhibition of protein kinase CK2 by [5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA)

Biochemical Journal ◽

10.1042/bj20030674 ◽

2003 ◽

Vol 374 (3) ◽

pp. 639-646 ◽

Cited By ~ 98

Author(s):

Stefania SARNO ◽

Erika De MOLINER ◽

Maria RUZZENE ◽

Mario A. PAGANO ◽

Roberto BATTISTUTTA ◽

...

Keyword(s):

Acetic Acid ◽

Protein Kinase ◽

Three Dimensional ◽

Binding Pocket ◽

Jurkat Cells ◽

Dependent Manner ◽

Phosphoinositide 3 Kinase ◽

Ck2 Inhibitors

IQA {[5-oxo-5,6-dihydro-indolo(1,2-a)quinazolin-7-yl]acetic acid} is a novel ATP/GTP site-directed inhibitor of CK2 (‘casein kinase 2’), a pleiotropic and constitutively active protein kinase whose activity is abnormally high in transformed cells. The Ki value of IQA (0.17 μM) is lower than those of other CK2 inhibitors reported so far. Tested at 10 μM concentration in the presence of 100 μM ATP, IQA almost suppresses CK2 activity in vitro, whereas it is ineffective or weakly effective on a panel of 44 protein kinases and on phosphoinositide 3-kinase. In comparison, other CK2 inhibitors, notably apigenin and quercetin, are more promiscuous. The in vivo efficacy of IQA has been assessed by using the fact that treatment of Jurkat cells with IQA inhibits endogenous CK2 in a dose-dependent manner. IQA has been co-crystallized with maize CK2α, which is >70% identical with its human homologue, and the structure of the complex has been determined at 1.68 Å (1 Å=0.1 nm) resolution. The inhibitor lies in the same plane occupied by the purine moiety of ATP with its more hydrophobic side facing the hinge region. Major contributions to the interaction are provided by hydrophobic forces and non-polar interactions involving the aromatic portion of the inhibitor and the hydrophobic residues surrounding the ATP-binding pocket, with special reference to the side chains of V53 (Val53), I66, M163 and I174. Consequently, mutants of human CK2α in which either V66 (the homologue of maize CK2α I66) or I174 is replaced by alanine are considerably less sensitive to IQA inhibition when compared with wild-type. These results provide new tools for deciphering the enigmatic role of CK2 in living cells and may pave the way for the development of drugs depending on CK2 activity.

Download Full-text

Hic-5 expression is a major indicator of cancer cell morphology, migration, and plasticity in three-dimensional matrices

Molecular Biology of the Cell ◽

10.1091/mbc.e18-02-0092 ◽

2018 ◽

Vol 29 (14) ◽

pp. 1704-1717 ◽

Cited By ~ 13

Author(s):

Anushree C. Gulvady ◽

Fatemeh Dubois ◽

Nicholas O. Deakin ◽

Gregory J. Goreczny ◽

Christopher E. Turner

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Cancer Cell ◽

Ectopic Expression ◽

Three Dimensional ◽

Cell Types ◽

Cell Phenotype ◽

In Vitro Invasiveness

The focal adhesion proteins Hic-5 and paxillin have been previously identified as key regulators of MDA-MB-231 breast cancer cell migration and morphologic mesenchymal-amoeboid plasticity in three-dimensional (3D) extracellular matrices (ECMs). However, their respective roles in other cancer cell types have not been evaluated. Herein, utilizing 3D cell–derived matrices and fibronectin-coated one-dimensional substrates, we show that across a variety of cancer cell lines, the level of Hic-5 expression serves as the major indicator of the cells primary morphology, plasticity, and in vitro invasiveness. Domain mapping studies reveal sites critical to the functions of both Hic-5 and paxillin in regulating phenotype, while ectopic expression of Hic-5 in cell lines with low endogenous levels of the protein is sufficient to induce a Rac1-dependent mesenchymal phenotype and, in turn, increase amoeboid-mesenchymal plasticity and invasion. We show that the activity of vinculin, when coupled to the expression of Hic-5 is required for the mesenchymal morphology in the 3D ECM. Taken together, our results identify Hic-5 as a critical modulator of tumor cell phenotype that could be utilized in predicting tumor cell migratory and invasive behavior in vivo.

Download Full-text